Kv Channel Activity Research Articles

Basal NAD(H) redox state permits hydrogen peroxide-induced mesenteric artery dilatation.

Smooth muscle voltage-gated K+ (Kv) channels in resistance arteries control vascular tone and contribute to the coupling of blood flow with local metabolic activity. Members of the Kv1 family are expressed in vascular smooth muscle and are modulated upon physiological elevation of local metabolites, including the glycolytic end-product l-lactate and superoxide-derived hydrogen peroxide (H2 O2 ). Here, we show that l-lactate elicits vasodilatation of small-diameter mesenteric arteries in a mechanism that requires lactate dehydrogenase (LDH). Using the inside-out configuration of the patch clamp technique, we show that increases in NADH that reflect LDH-mediated conversion of l-lactate to pyruvate directly stimulate the activity of single Kv1 channels and significantly enhance the sensitivity of Kv1 activity to H2 O2 . Consistent with these findings, H2 O2 -evoked vasodilatation was significantly greater in the presence of 10mM l-lactate relative to lactate-free conditions, yet was abolished in the presence of 10mM pyruvate, which shifts the LDH reaction towards the generation of NAD+ . Moreover, the enhancement of H2 O2 -induced vasodilatation was abolished in arteries from double transgenic mice with selective overexpression of the intracellular Kvβ1.1subunit in smooth muscle cells. Together, our results indicate that the Kvβ complex of native vascular Kv1 channels serves as a nodal effector for multiple redox signals to precisely control channel activity and vascular tone in the face of dynamic tissue-derived metabolic cues. KEY POINTS: Vasodilatation of mesenteric arteries by elevated external l-lactate requires its conversion by lactate dehydrogenase. Application of either NADH or H2 O2 potentiates single Kv channel currents in excised membrane patches from mesenteric artery smooth muscle cells. The binding of NADH enhances the stimulatory effects of H2 O2 on single Kv channel activity. The vasodilatory response to H2 O2 is differentially modified upon elevation of external l-lactate or pyruvate. The presence of l-lactate enhances the vasodilatory response to H2 O2 via the Kvβ subunit complex in smooth muscle.

Site and Mechanism of ML252 Inhibition of Kv7 Voltage-Gated Potassium Channels.

Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.

Corticotropin-releasing hormone neurons in the central nucleus of amygdala are required for chronic stress-induced hypertension.

Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension. Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs. CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.

Rapid and Robust Multi-Phenotypic Assay System for ALS Using Human iPS Cells with Mutations in Causative Genes.

Amyotrophic lateral sclerosis (ALS) is a major life-threatening disease caused by motor neuron degeneration. More effective treatments through drug discovery are urgently needed. Here, we established an effective high-throughput screening system using induced pluripotent stem cells (iPSCs). Using a Tet-On-dependent transcription factor expression system carried on the PiggyBac vector, motor neurons were efficiently and rapidly generated from iPSCs by a single-step induction method. Induced iPSC transcripts displayed characteristics similar to those of spinal cord neurons. iPSC-generated motor neurons carried a mutation in fused in sarcoma (FUS) and superoxide dismutase 1 (SOD1) genes and had abnormal protein accumulation corresponding to each mutation. Calcium imaging and multiple electrode array (MEA) recordings demonstrated that ALS neurons were abnormally hyperexcitable. Noticeably, protein accumulation and hyperexcitability were ameliorated by treatment with rapamycin (mTOR inhibitor) and retigabine (Kv7 channel activator), respectively. Furthermore, rapamycin suppressed ALS neuronal death and hyperexcitability, suggesting that protein aggregate clearance through the activation of autophagy effectively normalized activity and improved neuronal survival. Our culture system reproduced several ALS phenotypes, including protein accumulation, hyperexcitability, and neuronal death. This rapid and robust phenotypic screening system will likely facilitate the discovery of novel ALS therapeutics and stratified and personalized medicine for sporadic motor neuron diseases.

Neuron-oligodendrocyte potassium shuttling at nodes of Ranvier protects against inflammatory demyelination.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte-Kir4.1-deficient (OL-Kir4.1-deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

The Role of the Superoxide Radical in the Regulation of the K<sub>V</sub>-Channels Function in the Coronary Vessels Following Posttraumatic Stress Disorder

The purpose of the research was to study the contribution of the superoxide radical (\({\text{O}}_{2}^{{\centerdot - }}\)) to the mechanisms of the coronary KV-channels functional activity impairment in rats with posttraumatic stress disorder (PTSD). The study was performed on 117 outbred white male rats (Rattus, Muridae weighing 210–240 g). To reproduce the experimental analogue of PTSD, the modified model of “a predator presence imitation” was applied (contact with cats’ excrements for 10 days, 15 minutes daily). The PTSD development was confirmed by behavioral changes of affected animals in the “Open Field” test. The coronary vessels’ tone was studied on the isolated by the Langendorf’s method isotonically contracted hearts, which were perfused under constant flow with Krebs–Henseleit solution. The contribution of \({\text{O}}_{2}^{{\centerdot - }}\) to the coronary vascular tone regulation was studied by infusion of the superoxide radical “scavenger” Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) in the perfusion solution. The functional activity of KV-channels was assessed by the degree of increase in the coronary perfusion pressure (CPP) in response to 4-aminopyridine (4-AP), a blocker of KV-channels. To elucidate the role of \({\text{O}}_{2}^{{\centerdot - }}\) in the KV‑channels functional activity of coronary vessels, Tiron and 4-AP were infused in the perfusion solution. The concentration of diene conjugates (DCs), malondialdehyde (MDA), C‑reactive protein (C-RP) and interleukin 1β (IL-1β) as well as catalase (CAT) and superoxide dismutase (SOD) activity were detected in the blood serum of experimental animals. The CPP in the isolated rats’ hearts after PTSD was 30% lower at coronary flow rate 10 mL/min compared to the control group. Under the influence of 4-AP, CPP increased by 70% and by 24% in the “Control” and “PTSD” groups, respectively. Under influence of Tiron, the CPP in the “PTSD” group at coronary flow rate 10 mL/min was 52% lower than in the control. In the “PTSD + Tiron + 4-AP” group the CPP augmentation (71.5%) was comparable to that in the hearts of the “Control” group after 4-AP exposure. The IL-1β, C-RP, DCs and MDA concentration in the blood serum of rats with PTSD was 3, 1.6, 3.3 and 3.6 times higher than in the control rats’ blood serum, while SOD and CAT activity was by 27 and 59% lower, compared to control, respectively. In the course of the investigation, it was found that \({\text{O}}_{2}^{{\centerdot - }}\) overproduction due to oxidative stress might be an important mechanism of a poststressor “channelopathy”, which is characterized by the decreased functional activity of the KV-channels of coronary vessels following PTSD.

Voltage sensor targeted activation of Kv7 channels by QO-58.

Kv7 (KCNQ) voltage-gated potassium channels are key modulators of neuronal excitability and have been a promising target for developing novel drugs to treat epilepsy and pain. Drug discovery has focused on identifying small molecules that modulate channel function and understanding their mechanisms of action. Special attention has been given to Kv7 channel activators, which may have therapeutic benefits in epilepsy or pain. In this study, we compared the effects and mechanism of a candidate Kv7 activator, QO-58, which has been previously described but has an unclear site and mechanism of action. We compared QO-58 actions with other well-characterized activators that influence either the VSD (ICA-069673) or pore domain (ML213) of Kv7 channels. QO-58 exhibits strong subtype specificity for Kv7.2 over Kv7.3, similar to VSD-targeted activators. Additionally, QO-58 exhibits prominent state-dependent actions, as it requires channels to activate before binding, and dramatically decelerates closure of Kv7.2. We also tested the effects of multiple VSD mutations on QO-58 and ICA-069673 actions. Interestingly, we found that the Kv7.2[F168L] mutation, previously shown to abolish ICA-069673 sensitivity, does not markedly affect QO-58 mediated effects. Notably, Kv7.2[I134A] and Kv7.2[I173T] weaken the effects of ICA-069673, but are hypersensitive to QO-58. This finding may suggest that dimensions of the VSD binding pocket influence the potency of smaller compounds (e.g. ICA-069673) relative to bulkier ones (e.g. QO-58), despite overlap in their binding sites. Our findings highlight that key contacts underlying sensitivity to VSD-targeted activators may vary significantly depending on the chemical and steric features of the drug. We anticipate that further investigation of VSD-targeted activators will continue to clarify the complex pharmacophore of the VSD binding pocket in Kv7 channels.

Yield, cell composition, and function of islets isolated from different ages of neonatal pigs.

The yield, cell composition, and function of islets isolated from various ages of neonatal pigs were characterized using in vitro and in vivo experimental models. Islets from 7- and 10-day-old pigs showed significantly better function both in vitro and in vivo compared to islets from 3- and 5-day-old pigs however, the islet yield from 10-day-old pigs were significantly less than those obtained from the other pigs. Since islets from 3-day-old pigs were used in our previous studies and islets from 7-day-old pigs reversed diabetes more efficiently than islets from other groups, we further evaluated the function of these islets post-transplantation. B6 rag-/- mouse recipients of various numbers of islets from 7-day-old pigs achieved normoglycemia faster and showed significantly improved response to glucose challenge compared to the recipients of the same numbers of islets from 3-day-old pigs. These results are in line with the findings that islets from 7-day-old pigs showed reduced voltage-dependent K+ (Kv) channel activity and their ability to recover from post-hypoxia/reoxygenation stress. Despite more resident immune cells and immunogenic characteristics detected in islets from 7-day-old pigs compared to islets from 3-day-old pigs, the combination of anti-LFA-1 and anti-CD154 monoclonal antibodies are equally effective at preventing the rejection of islets from both age groups of pigs. Collectively, these results suggest that islets from various ages of neonatal pigs vary in yield, cellular composition, and function. Such parameters may be considered when defining the optimal pancreas donor for islet xenotransplantation studies.

Attenuation of Epileptogenesis and Cognitive Deficits by a Selective and Potent Kv7 Channel Opener in Rodent Models of Seizures.

Targeting neuronal Kv7 channels by pharmacological activation has been proven to be an attractive therapeutic strategy for epilepsy. Here, we show that activation of Kv7 channels by an opener SCR2682 dose-dependently reduces seizure activity and severity in rodent models of epilepsy induced by a GABAa receptor antagonist pentylenetetrazole (PTZ), maximal electroshock, and a glutamate receptor agonist kainic acid (KA). Electroencephalographic recordings of rat cerebral cortex confirm that SCR2682 also decreases epileptiform discharges in KA-induced seizures. Nissl and neuronal nuclei staining further demonstrates that SCR2682 also protects neurons from injury induced by KA. In Morris water maze navigation and Y-maze tests, SCR2682 improves PTZ- and KA-induced cognitive impairment. Taken together, our findings demonstrate that pharmacological activation of Kv7 by novel opener SCR2682 may hold promise for therapy of epilepsy with cognitive impairment. SIGNIFICANCE STATEMENT: A neuronal Kv7 channel opener SCR2682 attenuates epileptogenesis and seizure-induced cognitive impairment in rodent models of seizures, thus possessing a developmental potential for effective therapy of epilepsy with cognitive impairment.

Kv1 channel inactivation: Slow and slower.

JGP study suggests that Kv1 channels share a common mechanism of slow inactivation, but that some family members are less prone to inactivate than others.

Mutations within the selectivity filter reveal that Kv1 channels have distinct propensities to slow inactivate.

Voltage-activated potassium (Kv) channels open in response to membrane depolarization and subsequently inactivate through distinct mechanisms. For the model Shaker Kv channel from Drosophila, fast N-type inactivation is thought to occur by a mechanism involving blockade of the internal pore by the N-terminus, whereas slow C-type inactivation results from conformational changes in the ion selectivity filter in the external pore. Kv channel inactivation plays critical roles in shaping the action potential and regulating firing frequency, and has been implicated in a range of diseases including episodic ataxia and arrhythmias. Although structures of the closely related Shaker and Kv1.2 channels containing mutations that promote slow inactivation both support a mechanism involving dilation of the outer selectivity filter, mutations in the outer pores of these two Kv channels have been reported to have markedly distinct effects on slow inactivation, raising questions about the extent to which slow inactivation is related in both channels. In this study, we characterized the influence of a series of mutations within the external pore of Shaker and Kv1.2 channels and observed many distinct mutant phenotypes. We find that mutations at four positions near the selectivity filter promote inactivation less dramatically in Kv1.2 when compared to Shaker, and they identify one key variable position (T449 in Shaker and V381 in Kv1.2) underlying the different phenotypes in the two channels. Collectively, our results suggest that Kv1.2 is less prone to inactivate compared to Shaker, yet support a common mechanism of inactivation in the two channels.

The antidiabetic drug teneligliptin induces vasodilation via activation of PKG, Kv channels, and SERCA pumps in aortic smooth muscle

Diabetes mellitus (DM) is a metabolic disease closely related to cardiovascular disease. The dipeptidyl peptidase-4 inhibitor teneligliptin is used to treat DM and has recently been shown to have a cardiovascular protective effect against diseases such as hypertension and heart failure. The present study demonstrates the vasodilatory effect of teneligliptin using aortic rings pre-contracted with phenylephrine. Teneligliptin induced a vasodilatory effect in a dose-dependent manner, with and without endothelium. In addition, pretreatment with the nitric oxide synthase inhibitor L-NAME and small-conductance Ca2+-activated K+ channel inhibitor apamin did not alter the teneligliptin-induced vasodilatory effect. Although the adenylyl cyclase inhibitor SQ 22536 and protein kinase A (PKA) inhibitor KT 5720 did not modulate the vasodilatory effect of teneligliptin, the guanylyl cyclase inhibitor ODQ and protein kinase G (PKG) inhibitor KT 5823 effectively reduced the effect of teneligliptin. Similarly, pretreatment with the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (4-AP) also reduced teneligliptin-induced vasodilation. However, pretreatment with the inward rectifier K+ (Kir) channel inhibitor Ba2+, large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline, and ATP-sensitive K+ (KATP) channel inhibitor glibenclamide did not alter the vasodilatory effect of teneligliptin. Our data suggest that Kv7.X, but not Kv1.5 or Kv2.1, is one of the major Kv subtypes involved in teneligliptin-induced vasodilation. Furthermore, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin and CPA inhibited the vasodilation induced by teneligliptin. Our results suggest that teneligliptin-induced vasodilation occurs via activation of PKG, SERCA pumps and Kv channels, but not the PKA signaling pathway, other K+ channels, or endothelium.

Rosa damascena Miller essential oil relaxes rat trachea via KV channels, KATP channels, and BKCa channels

Aim/ObjectiveThis study aimed to investigate the effects of Rosa damascena Miller essential oil on rat tracheal smooth muscle contractility and the hypothesis that voltage-gated potassium (KV) channels, ATP-sensitive potassium (KATP) channels, and large-conductance calcium-activated potassium (BKCa) channels may have roles in these effects. MethodsIsometric contraction-relaxation responses of tracheal rings were measured with an isolated tissue bath model. The steady contraction was induced with both 10−5 M ACh and 60 mM KCl, and then the concentration-dependent responses of rose oil (0.1–100 µg/mL) were examined. The time-matched control (double distilled water) group was also formed. To evaluate the role of KV, KATP, and BKCa channels, tracheal rings were incubated with 4-AP (KV channel blocker), glibenclamide (KATP channel blocker), TEA (BKCa channel blocker), and iberiotoxin (selective BKCa channel blocker). Also, a vehicle control group was formed for dimethyl sulfoxide (DMSO). ResultsRose oil exerted the relaxant effects in tracheal rings pre-contracted with both ACh and KCl at concentrations of 1, 10, and 100 µg/mL (p < 0.05). Besides, KV channel blocker 4-AP, KATP channel blocker glibenclamide, and BKCa channel blockers TEA and iberiotoxin incubations significantly inhibited the rose oil-induced relaxation responses (p < 0.05). However, incubation of tissues with DMSO, glibenclamide solvent, for 10 min did not cause a significant change in the relaxation responses to rose oil (p > 0.05). ConclusionsIn conclusion, the first physiological findings were obtained regarding the functional relaxant effects of rose essential oil in rat trachea. The findings showed that rose oil induces bronchorelaxation in a concentration-dependent manner. Besides, this study is the first to report that rose oil-mediated bronchodilation responses are associated with the activity of KV, KATP, and BKCa channels. These results suggest that rose oil might be a useful agent in the treatment of abnormal bronchoconstriction-related diseases such as asthma and chronic obstructive pulmonary disease.

Beyond Retigabine: Design, Synthesis, and Pharmacological Characterization of a Potent and Chemically Stable Neuronal Kv7 Channel Activator with Anticonvulsant Activity.

Neuronal Kv7 channels represent important pharmacological targets for hyperexcitability disorders including epilepsy. Retigabine is the prototype Kv7 activator clinically approved for seizure treatment; however, severe side effects associated with long-term use have led to its market discontinuation. Building upon the recently described cryoEM structure of Kv7.2 complexed with retigabine and on previous structure–activity relationship studies, a small library of retigabine analogues has been designed, synthesized, and characterized for their Kv7 opening ability using both fluorescence- and electrophysiology-based assays. Among all tested compounds, 60 emerged as a potent and photochemically stable neuronal Kv7 channel activator. Compared to retigabine, compound 60 displayed a higher brain/plasma distribution ratio, a longer elimination half-life, and more potent and effective anticonvulsant effects in an acute seizure model in mice. Collectively, these data highlight compound 60 as a promising lead compound for the development of novel Kv7 activators for the treatment of hyperexcitability diseases.

HIV-Induced Hyperactivity of Striatal Neurons Is Associated with Dysfunction of Voltage-Gated Calcium and Potassium Channels at Middle Age.

Despite combination antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) occur in ~50% of people living with HIV (PLWH), which are associated with dysfunction of the corticostriatal pathway. The mechanism by which HIV alters the neuronal activity in the striatum is unknown. The goal of this study is to reveal the dysfunction of striatal neurons in the context of neuroHIV during aging. Using patch-clamping electrophysiology, we evaluated the functional activity of medium spiny neurons (MSNs), including firing, Ca2+ spikes mediated by voltage-gated Ca2+ channels (VGCCs), and K+ channel-mediated membrane excitability, in brain slices containing the dorsal striatum (a.k.a. the caudate-putamen) from 12-month-old (12mo) HIV-1 transgenic (HIV-1 Tg) rats. We also assessed the protein expression of voltage-gated Cav1.2/Cav1.3 L-type Ca2+ channels (L-channels), NMDA receptors (NMDAR, NR2B subunit), and GABAA receptors (GABAARs, β2,3 subunit) in the striatum. We found that MSNs had significantly increased firing in 12mo HIV-1 Tg rats compared to age-matched non-Tg control rats. Unexpectedly, Ca2+ spikes were significantly reduced, while Kv channel activity was increased, in MSNs of HIV-1 Tg rats compared to non-Tg ones. The reduced Ca2+ spikes were associated with an abnormally increased expression of a shorter, less functional Cav1.2 L-channel form, while there was no significant change in the expression of NR2Bs or GABAARs. Collectively, the present study initially reveals neuroHIV-induced dysfunction of striatal MSNs in 12mo-old (middle) rats, which is uncoupled from VGCC upregulation and reduced Kv activity (that we previously identified in younger HIV-1 Tg rats). Notably, such striatal dysfunction is also associated with HIV-induced hyperactivity/neurotoxicity of glutamatergic pyramidal neurons in the medial prefrontal cortex (mPFC) that send excitatory input to the striatum (demonstrated in our previous studies). Whether such MSN dysfunction is mediated by alterations in the functional activity instead of the expression of NR2b/GABAAR (or other subtypes) requires further investigation.

Changes of Pulmonary Microhemodynamics in Experimental Pulmonary Thromboembolism after Pretreatment with K-Channel Activators.

Changes in pulmonary microhemodynamics in response to pulmonary embolism under conditions of activation of KATP channels with nicorandil, Kv channels with dapagliflozin, and BKCa channels with Evans blue were studied on isolated rabbit lungs. Under conditions of activation of KATP and BKCa channels, the constrictor reactions of the pulmonary arterial vessels during embolization of the pulmonary artery were less pronounced than in the control. Activation of BKCa channels reduced constrictor reactions of the pulmonary venous vessels, while activation of KATP and Kv channels eliminates them. The shifts of the capillary filtration coefficient are determined to a greater extent by the pre-/postcapillary resistance ratio, than by changes of the endothelial permeability. Pretreatment with dapagliflozin led to a decrease in the capillary filtration coefficient. It was established, that nimesulide exhibits properties of a BKCa-channel activator.

Mechanisms Underlying C-type Inactivation in Kv Channels: Lessons From Structures of Human Kv1.3 and Fly Shaker-IR Channels.

Voltage-gated potassium (Kv) channels modulate the function of electrically-excitable and non-excitable cells by using several types of “gates” to regulate ion flow through the channels. An important gating mechanism, C-type inactivation, limits ion flow by transitioning Kv channels into a non-conducting inactivated state. Here, we highlight two recent papers, one on the human Kv1.3 channel and the second on the Drosophila Shaker Kv channel, that combined cryogenic electron microscopy and molecular dynamics simulation to define mechanisms underlying C-type inactivation. In both channels, the transition to the non-conducting inactivated conformation begins with the rupture of an intra-subunit hydrogen bond that fastens the selectivity filter to the pore helix. The freed filter swings outwards and gets tethered to an external residue. As a result, the extracellular end of the selectivity filter dilates and K+ permeation through the pore is impaired. Recovery from inactivation may entail a reversal of this process. Such a reversal, at least partially, is induced by the peptide dalazatide. Binding of dalazatide to external residues in Kv1.3 frees the filter to swing inwards. The extracellular end of the selectivity filter narrows allowing K+ to move in single file through the pore typical of conventional knock-on conduction. Inter-subunit hydrogen bonds that stabilize the outer pore in the dalazatide-bound structure are equivalent to those in open-conducting conformations of Kv channels. However, the intra-subunit bond that fastens the filter to the pore-helix is absent, suggesting an incomplete reversal of the process. These mechanisms define how Kv channels self-regulate the flow of K+ by changing the conformation of the selectivity filter.

Time‐limited alterations in cortical activity of a knock‐in mouse model of KCNQ2‐related developmental and epileptic encephalopathy

De novo missense variants in the KCNQ2 gene encoding the Kv7.2 subunit of voltage-gated potassium Kv7/M channels are the main cause of developmental and epileptic encephalopathy with neonatal onset. Although seizures usually resolve during development, cognitive/motor deficits persist. To gain a better understanding of the cellular mechanisms underlying network dysfunction and their progression over time, we investigated in vivo, using local field potential recordings of freely moving animals, and ex vivo in layers II/III and V of motor cortical slices, using patch-clamp recordings, the electrophysiological properties of pyramidal cells from a heterozygous knock-in mouse model carrying the Kv7.2 p.T274M pathogenic variant during neonatal, postweaning and juvenile developmental stages. We found that knock-in mice displayed spontaneous seizures preferentially at postweaning rather than at juvenile stages. At the cellular level, the variant led to a reduction in M ​​current density/conductance and to neuronal hyperexcitability. These alterations were observed during the neonatal period in pyramidal cells of layers II/III and during the postweaning stage in pyramidal cells of layer V. Moreover, there was an increase in the frequency of spontaneous network-driven events mediated by GABA receptors, suggesting that the excitability of interneurons was also increased. However, all these alterations were no longer observed in layers II/III and V of juvenile mice. Thus, our data indicate that the action of the variant is regulated developmentally. This raises the possibility that the age-related seizure remission observed in KCNQ2-related developmental and epileptic encephalopathy patients results from a time-limited alteration of Kv7 channel activity and neuronal excitability. KEY POINTS: The electrophysiological impact of the pathogenic c.821C>T mutation of the KCNQ2 gene (p.T274M variant in Kv7.2 subunit) related to developmental and epileptic encephalopathy has been analysed both in vivo and ex vivo in layers II/III and V of motor cortical slices from a knock-in mouse model during development at neonatal, postweaning and juvenile stages. Mcurrent density and conductance are decreased and the excitability of layer II/III pyramidal cells is increased in slices from neonatal and postweaning knock-in mice but not from juvenile knock-in mice. Mcurrent and excitability of layer V pyramidal cells are impacted in knock-in mice only at the postweaning stage. Spontaneous GABAergic network-driven events can be recorded until the postweaning stage, and their frequency is increased in layers II/III of the knock-in mice. Knock-in mice display spontaneous seizures preferentially at postweaning rather than at juvenile stages.

The inhibitory effects of pimozide, an antipsychotic drug, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells

Pimozide is an antipsychotic drug used to treat chronic psychosis, such as Tourette’s syndrome. Despite its widespread clinical use, pimozide can cause unexpected adverse effects, including arrhythmias. However, the adverse effects of pimozide on vascular K+ channels have not yet been determined. Therefore, we investigated the effects of pimozide on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Pimozide concentration-dependently inhibited the Kv currents with an IC50 value of 1.78 ± 0.17 μM and a Hill coefficient of 0.90 ± 0.05. The inhibitory effect on the Kv current by pimozide was highly voltage-dependent in the voltage range of Kv channel activation, and additive inhibition of the Kv current by pimozide was observed in the full activation voltage range. The decay rate of inactivation was significantly accelerated by pimozide. Pimozide shifted the inactivation curve to a more negative potential. The recovery time constant from inactivation increased in the presence of pimozide. Furthermore, pimozide-induced inhibition of the Kv current was augmented by applying train pulses. Although pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and the Kv7 subtype inhibitor linopirdine did not alter the degree of pimozide-induced inhibition of the Kv currents, pretreatment with the Kv1.5 channel inhibitor DPO-1 reduced the inhibitory effects of pimozide on Kv currents. Pimozide induced membrane depolarization. We conclude that pimozide inhibits Kv currents in voltage-, time-, and use (state)-dependent manners. Furthermore, the major Kv channel target of pimozide is the Kv1.5 channel.

Therapeutic Effects of Kv7 Channel Activator Retigabine against Seizures and Neurodegeneration in Kainic Acid-Induced Seizure Model

Therapeutic Effects of Kv7 Channel Activator Retigabine against Seizures and Neurodegeneration in Kainic Acid-Induced Seizure Model