Channel Inhibitor Research Articles

Long-term changes in transmembrane voltage after electroporation are governed by the interplay between nonselective leak current and ion channel activation

Electroporation causes a temporal increase in cell membrane permeability and leads to prolonged changes in transmembrane voltage (TMV) in both excitable and non-excitable cells. However, the mechanisms of these TMV changes remain to be fully elucidated. To this end, we monitored TMV over 30 min after exposing two different cell lines to a single 100 µs electroporation pulse using the FLIPR Membrane Potential dye. In CHO-K1 cells, which express very low levels of endogenous ion channels, membrane depolarization following pulse exposure could be explained by nonselective leak current, which persists until the membrane reseals, enabling the cells to recover their resting TMV. In U-87 MG cells, which express many different ion channels, we unexpectedly observed membrane hyperpolarization following the initial depolarization phase, but only at 33 °C and not at 25 °C. We developed a theoretical model, supported by experiments with ion channel inhibitors, which indicated that hyperpolarization could largely be attributed to the activation of calcium-activated potassium channels. Ion channel activation, coupled with changes in TMV and intracellular calcium, participates in various physiological processes, including cell proliferation, differentiation, migration, and apoptosis. Therefore, our study suggests that ion channels could present a potential target for influencing the biological response after electroporation.

Potassium channel TASK-5 forms functional heterodimers with TASK-1 and TASK-3 to break its silence

TASK-5 (KCNK15) belongs to the acid-sensitive subfamily of two-pore domain potassium (K2P) channels, which includes TASK-1 and TASK-3. TASK-5 stands out as K2P channel for which there is no functional data available, since it was reported in 2001 as non-functional and thus “silent”. Here we show that TASK-5 channels are indeed non-functional as homodimers, but are involved in the formation of functional channel complexes with TASK-1 and TASK-3. TASK-5 negatively modulates the surface expression of TASK channels, while the heteromeric TASK-5-containing channel complexes located at the plasma membrane are characterized by changes in single-channel conductance, Gq-coupled receptor-mediated channel inhibition, and sensitivity to TASK modulators. The unique pharmacology of TASK-1/TASK-5 heterodimers, affected by a common polymorphism in KCNK15, needs to be carefully considered in the future development of drugs targeting TASK channels. Our observations provide an access to study TASK-5 at the functional level, particularly in malignant cancers associated with KCNK15.

The Efficacy Of Continuous Non-Inhaled Intranasal Carbon Dioxide (CO2) In The Abortive Treatment Of Migraine: Results From Three Randomized, Double-Blinded, Placebo-Controlled Trials

Objective: Determining the efficacy, tolerability, and safety of continuous non-inhaled intranasal carbon dioxide (CO2) in the acute or abortive treatment of migraine. Background: Carbon dioxide gas infused in the nose and sinuses potentially blocks trigeminal nerve activation and subsequent neuropeptide release, probably through inhibition of voltage-gated channels. The pathogenesis of the migraine headache involves activation of the trigeminocervical system, which establishes the nociceptive innervation of the head through the trigeminal and occipital nerves. Methods: In three randomized, double-blinded, placebo-controlled trials, we randomized 350 subjects with migraine as defined by the International Headache Society (intent-to-treat population). We determined tolerability and safety in the 317 subjects who we exposed to treatment up to 24 hours post-dose (modified intent-to-treat population). We determined efficacy in the modified intent-to-treat and per-protocol populations at 15 minutes, half, one, two, and twenty-four hours post-dose in terms of pain freedom, freedom from headache-associated symptoms, and headache relief, treating migraine headaches of mild, moderate, or severe intensity. Results: We did not observe clinically significant systemic effects, either on blood carbon dioxide pressure or on the cardiovascular or respiratory system. No serious or clinically significant adverse events occurred and those reported were predominantly local, transient, and short-lived. Headache intensity strongly and in a negative way determined the efficacy of treatment with carbon dioxide but not with placebo, and even when placebo consisted of no gas, blinding seemed adequate. Conclusions: Continuous non-inhaled intranasal carbon dioxide seems safe, relatively well tolerated, and moderately effective for the acute or abortive treatment of migraine.

Vasorelaxant Effect and Blood Pressure Reduction Potential of Pitaya Juice Concentrate (Stenocereus huastecorum) Associated with Calcium Channel Blockade.

Arterial hypertension is a highly prevalent chronic disease worldwide, with several etiologies and treatments that may eventually have side effects or result in patients developing tolerance. There is growing interest in traditional medicine and functional foods to isolate biomolecules that could be useful as coadjuvants for treating several aliments. Pitaya, a desert fruit endemic in Mexico, is a rich source of bioactive molecules (betalains and phenolic compounds). In this work, the vasorelaxation properties of pitaya juice concentrate and fraction one were investigated using aortic and mesenteric rings from rats. The incubation of rings with pitaya juice concentrate or fraction one induced significant vasorelaxation, independent of the endothelium, and showed resistance to potassium channel blockers. This vasorelaxation was associated with the transmembrane influx of extracellular calcium through the vascular smooth muscle cells, with an inhibitory effect on the voltage-dependent calcium channel currents. Also, 400 mg/mL of pitaya juice concentrate in spontaneous hypertensive rats reduced their blood pressure for 48 h. Phytochemical analyses showed that the primary compounds in F1 were glycosidic in nature, and could be a complex mixture of disaccharides, dimeric disaccharides, or even tetrasaccharides. The glycosidic compounds found in F1 primarily contributed to vasodilatation, establishing a voltage-dependent calcium channel inhibition as a possible molecular target.

Agrimonia coreana Extract Exerts Its Therapeutic Effect through CRAC Channel Inhibition for Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (ACext) can be used in treating AD-related dermatologic symptoms. ACext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon ACext treatment. In a mouse model of AD, ACext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD.

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

Investigating the cardiotoxicity of N-n-butyl haloperidol iodide: Inhibition mechanisms on hERG channels

The human Ether-à-go-go-Related Gene (hERG) encodes a protein responsible for forming the alpha subunit of the IKr channel, which plays a crucial role in cardiac repolarization. The proper functioning of hERG channels is paramount in maintaining a normal cardiac rhythm. Inhibition of these channels can result in the prolongation of the QT interval and potentially life-threatening arrhythmias. Cardiotoxicity is a primary concern in the field of drug development. N-n-Butyl haloperidol iodide (F2), a derivative of haloperidol, has been investigated for its therapeutic potential. However, the impact of this compound on cardiac toxicity, specifically on hERG channels, remains uncertain. This study employs computational and experimental methodologies to examine the inhibitory mechanisms of F2 on hERG channels. Molecular docking and molecular dynamics simulations commonly used techniques in computational biology to predict protein-ligand complexes' binding interactions and stability. In the context of the F2-hERG complex, these methods can provide valuable insights into the potential binding modes and strength of interaction between F2 and the hERG protein. On the other hand, electrophysiological assays are experimental techniques used to characterize the extent and nature of hERG channel inhibition caused by various compounds. By measuring the electrical activity of the hERG channel in response to different stimuli, these assays can provide important information about the functional effects of ligand binding to the channel. The study's key findings indicate that F2 interacts with the hERG channel by forming hydrogen bonding, π-cation interactions, and hydrophobic forces. This interaction leads to the inhibition of hERG currents in a concentration-dependent manner, with an IC50 of 3.75 μM. The results presented in this study demonstrate the potential cardiotoxicity of F2 and underscore the significance of considering hERG channel interactions during its clinical development. This study aims to provide comprehensive insights into the interaction between F2 and hERG, which will may guid us in the safe use of F2 and in the development of new derivatives with high efficiency while low toxicity.

Toxicity of Bromo-DragonFLY as a New Psychoactive Substance: Application of In Silico Methods for the Prediction of Key Toxicological Parameters Important to Clinical and Forensic Toxicology.

Bromo-DragonFLY is a synthetic new psychoactive substance (NPS) that has gained attention due to its powerful and long-lasting hallucinogenic effects, legal status, and widespread availability. This study aimed to use various in silico toxicology methods to predict key toxicological parameters for Bromo-DragonFLY, including acute toxicity (LD50), genotoxicity, cardiotoxicity, health effects, and the potential for endocrine disruption. The results indicate significant acute toxicity with noticeable variations across different species, a low likelihood of genotoxic potential suggesting potential DNA damage, and a notable risk of cardiotoxicity associated with inhibition of the hERG channel. Evaluation of endocrine disruption suggests a low probability of Bromo-DragonFLY interacting with the estrogen receptor α (ER-α), indicating minimal estrogenic activity. These insights from in silico investigations are important for advancing our understanding of this NPS in forensic and clinical toxicology. These initial toxicological examinations establish a foundation for future research efforts and contribute to developing risk assessment and management strategies for using and misusing NPS.

Label-free, non-contact determination of resting membrane potential using dielectrophoresis

Measurement of cellular resting membrane potential (RMP) is important in understanding ion channels and their role in regulation of cell function across a wide range of cell types. However, methods available for the measurement of RMP (including patch clamp, microelectrodes, and potential-sensitive fluorophores) are expensive, slow, open to operator bias, and often result in cell destruction. We present non-contact, label-free membrane potential estimation which uses dielectrophoresis to determine the cytoplasm conductivity slope as a function of medium conductivity. By comparing this to patch clamp data available in the literature, we have demonstratet the accuracy of this approach using seven different cell types, including primary suspension cells (red blood cells, platelets), cultured suspension cells (THP-1), primary adherent cells (chondrocytes, human umbilical mesenchymal stem cells), and adherent (HeLa) and suspension (Jurkat) cancer cell lines. Analysis of the effect of ion channel inhibitors suggests the effects of pharmaceutical agents (TEA on HeLa; DMSO and neuraminidase on red blood cells) can also be measured. Comparison with published values of membrane potential suggest that the differences between our estimates and values recorded by patch clamp are accurate to within published margins of error. The method is low-cost, non-destructive, operator-independent and label-free, and has previously been shown to allow cells to be recovered after measurement.

SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism.

A major issue in Chronic Myeloid Leukemia (CML) is the persistence of quiescent leukemia stem cells (LSCs) in the hematopoietic niche under tyrosine kinase inhibitor (TKI) treatment. Here, using CFSE sorting, we show that low-proliferating CD34+ cells from CML patients in 3D co-culture hide under HS27A stromal cells during TKI treatment-a behavior less observed in untreated cells. Under the same conditions, Ba/F3p210 cells lose their spontaneous motility. In CML CD34+ and Ba/F3p210 cells, while Rac1 is completely inhibited by TKI, RhoA remains activated but is unable to signal to ROCK. Co-incubation of Ba/F3p210 cells with TKI, SKF-96365 (a calcium channel inhibitor), and EGF restores myosin II activation and amoeboid motility to levels comparable to untreated cells, sustaining the activation of ROCK. In CFSE+ CD34+ cells containing quiescent leukemic stem cells, co-incubation of TKI with SKF-96365 induced the expulsion of these cells from the HS27A niche. This study underscores the role of RhoA in LSC behavior under TKI treatment and suggests that SKF-96365 could remobilize quiescent CML LSCs through reactivation of the RhoA/ROCK pathway.

Vasorelaxant mechanisms of the antidiabetic anagliptin in rabbit aorta: roles of Kv channels and SERCA pump.

The present study investigated the vasorelaxant mechanisms of an oral antidiabetic drug, anagliptin, using phenylephrine (Phe)-induced pre-contracted rabbit aortic rings. Arterial tone measurement was performed in rabbit thoracic aortic rings. Anagliptin induced vasorelaxation in a dose-dependent manner. Pre-treatment with the classical voltagedependent K+ (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly decreased the vasorelaxant effect of anagliptin, whereas pre-treatment with the inwardly rectifying K+ (Kir) channel inhibitor Ba2+, the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, and the large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline did not attenuate the vasorelaxant effect. Furthermore, the vasorelaxant response of anagliptin was effectively inhibited by pre-treatment with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid. Neither cAMP/protein kinase A (PKA)-related signaling pathway inhibitors (adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor KT 5720) nor cGMP/protein kinase G (PKG)-related signaling pathway inhibitors (guanylyl cyclase inhibitor ODQ and PKG inhibitor KT 5823) reduced the vasorelaxant effect of anagliptin. Similarly, the anagliptin-induced vasorelaxation was independent of the endothelium. Based on these results, we suggest that anagliptin-induced vasorelaxation in rabbit aortic smooth muscle occurs by activating Kv channels and the SERCA pump, independent of other vascular K+ channels, cAMP/PKA- or cGMP/PKG-related signaling pathways, and the endothelium.

Inhibition of Cutaneous TRPV3 Channels by Natural Caffeic Acid for the Alleviation of Skin Inflammation.

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC50 values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors.

The effects of Cl− channel inhibitors and pyrethroid insecticides on calcium-activated chloride channels in neurons of Helicoverpa armigera

TMEM16A, a member of the Transmembrane protein 16 family, serves as the molecular basis for calcium activated chloride channels (CaCCs). We use RT-PCR to demonstrate the expression of TMEM16A in the neurons of Helicoverpa armigera, and record the CaCCs current of acute isolated neurons of H. armigera for the first time using patch clamp technology. In order to screen effective inhibitors of calcium-activated chloride channels, the inhibitory effects of four chloride channel inhibitors, CaCCinh-A01, NPPB, DIDS, and SITS, on CaCCs were compared. The inhibitory effects of the four inhibitors on the outward current of CaCCs were CaCCinh-A01 (10 μM, 56.31 %), NPPB (200 μM, 43.69 %), SITS (1 mM, 12.41 %) and DIDS (1 mM, 13.29 %). Among these inhibitors, CaCCinh-A01 demonstrated the highest efficacy as a blocker. To further explore whether calcium channel proteins can serve as potential targets of pyrethroids, we compared the effects of (type I) tefluthrin and (type II) deltamethrin on CaCCs. 10 μM and 100 μM tefluthrin can stimulate a large tail current in CaCCs, prolonging their deactivation time by 10.44 ms and 31.49 ms, and the V0.5 shifted in the hyperpolarization by 2–8 mV. Then, deltamethrin had no obvious effect on the deactivation and activation of CaCCs. Therefore, CaCCs of H. armigera can be used as a potential target of pyrethroids, but type I and type II pyrethroids have different effects on CaCCs.

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

A developmental neurotoxicity adverse outcome pathway (DNT-AOP) with voltage gate sodium channel (VGSC) inhibition as a molecular initiating event (MiE).

The adverse outcome pathway (AOP) framework serves as a practical tool for organising scientific knowledge that can be used to infer cause-effect relationships between stressor events and toxicity outcomes in intact organisms. However, a major challenge in the broader application of the AOP concept within regulatory toxicology is the development of a robust AOPs that can withstand peer review and acceptance. This is mainly due to the considerable amount of work required to substantiate the modular units of a complete AOP, which can take years from inception to completion. The methodology used here consisted of an initial assessment of a single chemical hazard using the Integrated Approach to Testing and Assessment (IATA) framework. An evidence-based approach was then used to gather empirical evidence combining systematic literature review methods with expert knowledge to ensure the effectiveness of the AOP development methodology. The structured framework used assured transparency, objectivity and comprehensiveness, and included expert knowledge elicitation for the evaluation of key event relationships (KERs). This stepwise approach led to the development of an AOP that begins with binding of chemicals to Voltage Gate Sodium Channels (VGSC/Nav) during mammalian development leading to adverse consequences in neurodevelopment evidenced as deficits in cognitive functions. Disruption of the formation of precise neural circuits by alterations in VGSC kinetics during the perinatal stages of brain development may also underlie neurodevelopmental disorders. Gaps in our understanding include the specific critical developmental windows and the quantitative relationship of binding to VGSC and subsequent disruption and cognitive function. Despite the limited quantitative information at all KER levels, regulatory applications of this AOP for DNT assessment have been identified.

Diversely evolved xibalbin variants from remipede venom inhibit potassium channels and activate PKA-II and Erk1/2 signaling

BackgroundThe identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold.ResultsOur screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses.ConclusionsOur functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.

Acute Metabolic Stress Induces Lymphatic Dysfunction Through KATP Channel Activation.

Lymphatic dysfunction is an underlying component of multiple metabolic diseases, including diabetes, obesity, and metabolic syndrome. We investigated the roles of KATP channels in lymphatic contractile dysfunction in response to acute metabolic stress induced by inhibition of the mitochondrial electron transport chain. Ex vivo popliteal lymphatic vessels from mice were exposed to the electron transport chain inhibitors antimycin A and rotenone, or the oxidative phosphorylation inhibitor/protonophore, CCCP. Each inhibitor led to a significant reduction in the frequency of spontaneous lymphatic contractions and calculated pump flow, without a significant change in contraction amplitude. Contraction frequency was restored by the KATP channel inhibitor, glibenclamide. Lymphatic vessels from mice with global Kir6.1 deficiency or expressing a smooth muscle-specific dominant negative Kir6.1 channel were resistant to inhibition. Antimycin A inhibited the spontaneous action potentials generated in lymphatic muscle and this effect was reversed by glibenclamide, confirming the role of KATP channels. Antimycin A, but not rotenone or CCCP, increased dihydrorhodamine fluorescence in lymphatic muscle, indicating ROS production. Pretreatment with tiron or catalase prevented the effect of antimycin A on wild-type lymphatic vessels, consistent with its action being mediated by ROS. Our results support the conclusion that KATP channels in lymphatic muscle can be directly activated by reduced mitochondrial ATP production or ROS generation, consequent to acute metabolic stress, leading to contractile dysfunction through inhibition of the ionic pacemaker controlling spontaneous lymphatic contractions. We propose that a similar activation of KATP channels contributes to lymphatic dysfunction in metabolic disease.

ATP-sensitive potassium channel opener, Nicorandil, inhibits NF-κB/AIM2/GSDMD pathway activation to protect against neuroinflammation in ischemic stroke

The absent in melanoma 2 (AIM2) inflammasome contributes to ischemic brain injury by inducing cell pyroptosis and inflammatory responses. Our research group has previously demonstrated that ATP-sensitive potassium channels (KATP channels) openers can modulate neuronal synaptic plasticity post-ischemic stroke for neuroprotection. However, the specific mechanisms of KATP channels in the inflammatory response following ischemic stroke remain unclear. Here, we assessed cellular damage by observing changes in BV-2 morphology and viability. 2,3,5-Triphenyl tetrazolium chloride (TTC) staining, mNSS scoring, Nissl staining, and TdT-mediated dUTP nick end labeling (TUNEL) staining were used to evaluate behavioral deficits, brain injury severity, and neuronal damage in mice subjected to middle cerebral artery occlusion (MCAO). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to measure cell pyroptosis and nuclear factor-kappaB (NF-κB) activation in vivo and in vitro. We observed that AIM2 protein expression was upregulated and localized within the cytoplasm of BV-2 cells. Notably, low-dose Nicorandil treatment reduced inflammatory cytokine secretion and pyroptosis-related protein expression, including AIM2, cleaved cysteinyl aspartate-specific protease-1 (cleaved caspase-1), and Gasdermin D N-terminal (GSDMD-NT). Further investigations revealed that the KATP channel inhibitor 5-HD upregulated p-NF-κB p65, NF-κB p65, and p-IκBα expression, reversing Nicorandil's neuroprotective effect in vivo. In summary, our results suggest that Nicorandil may serve as a potential therapeutic option for ischemic stroke. Targeting AIM2 and NF-κB represents effective strategies for inhibiting neuroinflammation.

Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785.

Voltage-gated K+ channels play central roles in human physiology, both in health and disease. A repertoire of inhibitors that are both potent and specific would therefore be of great value, not only as pharmacological agents but also as research tools. The small molecule RY785 has been described as particularly promising in this regard, as it selectively inhibits channels in the Kv2 subfamily with high potency. Kv2 channels are expressed in multiple cell types in humans, and are of particular importance for neuronal function. The mechanism of action of RY785 has not yet been determined at the molecular level, but functional studies indicate it differs from that of less specific inhibitors, such as quaternary-ammonium compounds or aminopyridines; RY785 is distinct also in that it is electroneutral. To examine this mechanism at the single-molecule level, we have carried out a series of all-atom molecular dynamics simulations based on the experimental structure of the Kv2.1 channel in the activated, open state. First, we report a 25-microsecond trajectory calculated in the absence of any inhibitor, under an applied voltage of 100 mV, which demonstrates outward K+ flow under simulation conditions at rates comparable to experimental measurements. Additional simulations in which either RY785 or tetraethylammonium (TEA) is introduced in solution show both inhibitors spontaneously enter the channel through the cytoplasmic gate, with distinct effects. In agreement with prior structural studies, we observe that TEA binds to a site adjacent to the selectivity filter, on the pore axis, thereby blocking the flow of K+ ions. RY785, by contrast, binds to the channel walls, off-axis, and allows K+ flow while the cytoplasmic gate remains open. The observed mode of RY785 binding, however, indicates that its mechanism of action is to stabilize and occlude a semi-open state of the gate, by bridging hydrophobic protein-protein interactions therein; this hypothesis would explain the puzzling experimental observation that RY785 recognition influences the gating currents generated by the voltage sensors, 3 nm away.

The TWIK-related acid sensitive potassium 3 (TASK-3) channel contributes to the different effects of anesthetics on the growth and metastasis of ovarian cancer cells

Different anesthetics exert different effects on the long-term outcomes of various cancers. The TWIK-related acid sensitive potassium 3 (TASK-3) channel is an important target of anesthetics and is upregulated in various cancers. However, the role and underlying mechanism of TASK-3 channel in the effects of anesthetics on ovarian cancer remain unknown. Here, we tested whether the TASK-3 channel contributes to the effects of anesthetics on ovarian cancers. We found that the TASK-3 channel was overexpressed in human ovarian cancer and ovarian cancer cell lines. Clinically relevant concentrations of lidocaine, as a TASK-3 channel inhibitor, exert inhibitory effects on tumor growth and metastasis of ovarian cancer cells in vitro and in vivo, whereas the TASK-3 channel potent activator sevoflurane had protumor effects and propofol had no significant effects on tumor growth and metastasis of ovarian cancer. Knockdown of the TASK-3 channel by TASK-3 shRNA attenuated the effects of lidocaine and sevoflurane. Moreover, mitochondrial TASK-3 channel contributes to the effects of lidocaine and sevoflurane on the mitochondrial functions of ovarian cancer. Taken together, the TASK-3 channel, especially the mitochondrial TASK-3 (MitoTASK-3) channel, is a molecular substrate for the effects of lidocaine and sevoflurane on the tumor growth and metastasis of ovarian cancer.