Activation Of Potassium Channels Research Articles

Molecular mechanism of GIRK2 channel gating modulated by cholesteryl hemisuccinate

Cholesterol, an essential lipid of cell membranes, regulates G protein-gated inwardly rectifying potassium (GIRK) channel activity. Previous studies have shown that cholesterol activates GIRK2 homotetrameric channels, which are expressed in dopaminergic neurons of the brain. Deletion of GIRK2 channels affects both GIRK2 homo- and heterotetrames and can lead to abnormal neuronal excitability, including conditions such as epilepsy and addiction. A 3.5 Å cryo-EM structure of GIRK2 in complex with CHS (cholesteryl hemisuccinate) and PIP2 (phosphatidylinositol 4,5-bisphosphate) has been solved. This structure provides the opportunity to study GIRK2 channel gating dynamics regulated by cholesterol using gating molecular dynamics (GMD) simulations. In the present study, we conducted microsecond-long GMD simulations on the GIRK2 channel in its APO, PIP2, and PIP2/CHS bound states, followed by systematic analysis to gain molecular insights into how CHS modulates GIRK2 channel gating. We found that CHS binding facilitates GIRK2 channel opening, with 43 K+ ion permeation events observed, compared to 0 and 2 K+ ion permeation events for GIRK2-APO and GIRK2/PIP2, respectively. Binding of CHS to the GIRK2 channel enhances PIP2 and channel interactions, which is consistent with previous experimental results. The negatively charged PIP2 alters the internal electrostatic potential field in the channel and lowers the negative free energy barrier for K+ ion permeation.

Exploring the bioactive ingredients of three traditional Chinese medicine formulas against age-related hearing loss through network pharmacology and experimental validation.

Traditional Chinese medicine (TCM) formulas, including the Er-Long-Zuo-Ci pill, Tong-Qiao-Er-Long pill, and Er-Long pill, have long been utilized in China for managing age-related hearing loss (ARHL). However, the specific bioactive compounds, pharmacological targets, and underlying mechanisms remain elusive. This study aims to find the shared bioactive ingredients among these three formulas, uncover the molecular pathways they regulate, and identify potential therapeutic targets for ARHL. Furthermore, it seeks to validate the efficacy of these major components through both in vivo and in vitro experiments. Common bioactive ingredients were extracted from the TCMSP database, and their putative target proteins were predicted using the Swiss Target Prediction database. ARHL-related target proteins were collected from GeneCards and OMIM databases. Our approach involved constructing drug-target networks and drug-disease-specific protein-protein interaction networks and conducting clustering, topological property analyses, and functional annotation through GO and KEGG enrichment analysis. Molecular docking analysis was utilized to delineate interaction mechanisms between major bioactive ingredients and key target proteins. Finally, in vivo and in vitro experiments involving ABR recording, immunofluorescent staining, HE staining, and quantitative PCR were conducted to validate the treatment effects of flavonoids on the declining auditory function in DBA/2J mice. We identified 11 common chemical compounds across the three formulas and their associated 276 putative targets. Additionally, 3350 ARHL-related targets were compiled. As an intersection of the putative targets of the common compounds and ARHL-related proteins, 145 shared targets were determined. Functional enrichment analysis indicated that these compounds may modulate various biological processes, including cell proliferation, apoptosis, inflammatory response, and synaptic connections. Notably, potential targets such as TNFα, MAPK1, SRC, AKT, EGFR, ESR1, and AR were implicated. Flavonoids emerged as major bioactive components against ARHL based on target numbers, with molecular docking demonstrating diverse interaction models between these flavonoids and protein targets. Furthermore, baicalin could mitigate the age-related cochlear damage and hearing loss of DBA/2J mice through its multi-target and multi-pathway mechanism, involving anti-inflammation, modulation of sex hormone-related pathways, and activation of potassium channels. This study offers an integrated network pharmacology approach, validated by in vivo and in vitro experiments, shedding light on the potential mechanisms, major active components, and therapeutic targets of TCM formulas for treating ARHL.

A novel KCNC3 gene variant in the voltage-dependent Kv3.3 channel in an atypical form of SCA13 with dominant central vertigo.

Potassium channel mutations play an important role in neurological diseases, such as spinocerebellar ataxia (SCA). SCA is a heterogeneous autosomal-dominant neurodegenerative disorder with multiple sub-entities, such as SCA13, which is characterized by mutations in the voltage-gated potassium channel Kv3.3 (KCNC3). In this study, we present a rare and atypical case of SCA13 with a predominant episodic central rotational vertigo, while the patient suffered only from mild progressive cerebellar symptoms, such as dysarthria, ataxia of gait and stand, and recently a cognitive impairment. In this patient, we identified a heterozygous variant in KCNC3 (c.2023G > A, p.Glu675Lys) by next-generation sequencing. This Kv3.3E675K variant was studied using voltage-clamp recordings in Xenopus oocytes. While typical SCA13 variants are dominant-negative, show shifts in the voltage-dependence of activation or an altered TBK1 regulation, the Kv3.3E675K variant caused only a reduction in current amplitude and a more pronounced cumulative inactivation. Thus, the differences to phenotypes observed in patients with classical SCA13 mutations may be related to the mechanism of the observed Kv3.3 loss-of-function. Treatment of our patient with riluzole, a drug that is known to also activate potassium channels, turned out to be partly beneficial. Strikingly, we found that the Kv3.3 and Kv3.3E675K inactivation and the frequency-dependent cumulative inactivation was antagonized by increased extracellular potassium levels. Thus, and most importantly, carefully elevated plasma potassium levels in the physiological range, or novel drugs attenuating Kv3.3 inactivation might provide novel therapeutic approaches to rescue potassium currents of SCA13 variants per se. In addition, our findings broaden the phenotypic spectrum of Kv3.3 variants, expanding it to atypical phenotypes of Kv3.3-associated neurological disorders.

Nicorandil antiallodynic activity in a model of neuropathic pain is associated with the activation of ATP-dependent potassium channels and opioidergic pathways, and reduced production of cytokines and neutrophils recruitment in paw, sciatic nerve, and dorsal root ganglia.

Recently, we demonstrated that nicorandil inhibits mechanical allodynia induced by paclitaxel. In the present study, we evaluated the effect induced by nicorandil in a model of neuropathic pain induced by chronic constriction injury (CCI) in mice. We also investigated putative mechanisms underlying such an effect. CCI was induced by three ligatures of the left sciatic nerve. Mechanical allodynia was evaluated by measuring the paw withdrawal threshold with an electronic von Frey apparatus. Concentrations of cytokines and myeloperoxidase activity were determined in the paw tissue, sciatic nerve, and dorsal root ganglia (DRG). Oral administration of two doses of nicorandil (150mg/kg po), but not equimolar doses of nicotinamide or nicotinic acid, attenuated mechanical allodynia induced by CCI. Nicorandil activity was reduced by previous administration of glibenclamide (40mg/kg) or naltrexone (5mg/kg or 10mg/kg). Two doses of nicorandil (150mg/kg, po) reduced tumor necrosis factor-α, interleukin-1β and interleukin-6, but not CXCL-1, concentrations in the paw tissue of CCI mice. Two doses of nicorandil (150mg/kg, po) reduced concentrations of all these mediators in the sciatic nerve and DRG. Two doses of nicorandil (150mg/kg, po) also reduced the myeloperoxidase activity in the paw tissue, sciatic nerve, and DRG. Nicorandil exhibits antiallodynic activity in a model of neuropathic pain induced by CCI. Inhibition of cytokines production and reduction of neutrophils recruitment in paw tissue, sciatic nerve, and DRG as well as activation of ATP-dependent potassium channels and opioidergic pathways, underlie nicorandil antiallodynic activity.

Discovery of E0199: A novel compound targeting both peripheral NaV and KV7 channels to alleviate neuropathic pain

This research study focuses on addressing the limitations of current neuropathic pain (NP) treatments by developing a novel dual-target modulator, E0199, targeting both NaV1.7, NaV1.8, and NaV1.9 and KV7 channels, a crucial regulator in controlling NP symptoms. The objective of the study was to synthesize a compound capable of modulating these channels to alleviate NP. Through an experimental design involving both in vitro and in vivo methods, E0199 was tested for its efficacy on ion channels and its therapeutic potential in a chronic constriction injury (CCI) mouse model. The results demonstrated that E0199 significantly inhibited NaV1.7, NaV1.8, and NaV1.9 channels with a particularly low half maximal inhibitory concentration (IC50) for NaV1.9 by promoting sodium channel inactivation, and also effectively increased KV7.2/7.3, KV7.2, and KV7.5 channels, excluding KV7.1 by promoting potassium channel activation. This dual action significantly reduced the excitability of dorsal root ganglion neurons and alleviated pain hypersensitivity in mice at low doses, indicating a potent analgesic effect without affecting heart and skeletal muscle ion channels critically. The safety of E0199 was supported by neurobehavioral evaluations. Conclusively, E0199 represents a ground-breaking approach in NP treatment, showcasing the potential of dual-target small-molecule compounds in providing a more effective and safe therapeutic option for NP. This study introduces a promising direction for the future development of NP therapeutics.

Repositioning pinacidil and its anticonvulsant and anxiolytic properties in murine models

Epilepsy, frequently comorbid with anxiety, is a prevalent neurological disorder. Available drugs often have side effects that hinder adherence, creating a need for new treatments. Potassium channel activators have emerged as promising candidates for treating both epilepsy and anxiety. This study aimed to evaluate the potential anticonvulsant and anxiolytic effects of pinacidil, an ATP-sensitive potassium channel activator used as antihypertensive, in rats. Our results indicate that pinacidil at 10 mg/kg (i.p.) fully protected animals from seizures induced by pentylenetetrazol (PTZ) and provided 85.7%, 100% and 100% protection against pilocarpine-induced seizures at 2.5, 5 and 10 mg/kg (i.p.), respectively. Although the 2.5 and 5 mg/kg (i.p) doses did not significantly protect the animals from PTZ-induced seizures, they did significantly increase the latency to the first seizure. Pinacidil also demonstrated mild anxiolytic activity, particularly at 10 mg/kg (i.p), evidenced by increased time spent in the open or illuminated areas of the Elevated Plus Maze (EPM) and Light-Dark Box (LDB) and increased exploratory activity in the Open Filed, EPM and LDB. Pinacidil did not affect locomotor performance, supporting its genuine anticonvulsant effects. This study holds significant medical and pharmaceutical value by characterizing pinacidil’s anticonvulsant and anxiolytic effects and highlighting its potential for therapeutic repositioning.

New Application of an Old Drug: Anti-Diabetic Properties of Phloroglucinol.

Phloroglucinol (PHG), an analgesic and spasmolytic drug, shows promise in preventing high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and insulin resistance. In Wistar rats, 10 weeks of PHG treatment did not prevent HFD-induced weight gain but significantly mitigated fasting hyperglycemia, impaired insulin responses, and liver steatosis. This protective effect was not linked to hepatic lipogenesis or AMP-activated protein kinase (AMPK) activation. Instead, PHG improved mitochondrial function by reducing oxidative stress, enhancing ATP production, and increasing anti-oxidant enzyme activity. PHG also relaxed gastric smooth muscles via potassium channel activation and nitric oxide (NO) signaling, potentially delaying gastric emptying. A pilot intervention in pre-diabetic men confirmed PHG's efficacy in improving postprandial glycemic control and altering lipid metabolism. These findings suggest PHG as a potential therapeutic for NAFLD and insulin resistance, acting through mechanisms involving mitochondrial protection, anti-oxidant activity, and gastric motility modulation. Further clinical evaluation is warranted to explore PHG's full therapeutic potential.

Exploring new horizons: angiotensin II, angiotensin II type 1 receptor, and renal outer medullary potassium channel interaction in distal convoluted tubule.

This study investigates angiotensin II (Ang II)'s regulatory mechanism on renal outer medullary potassium channel (ROMK) activity in the distal convoluted tubule (DCT) during low potassium intake, focusing on the Janus kinase 2 (JAK2) pathway activation mediated by the Ang II type 1 receptor (AT1R). Utilizing a low potassium diet mouse model, various methods including patch clamping, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining were applied to analyze ROMK channel activity and the expression of related proteins. The findings reveal that Ang II inhibits ROMK activity in the DCT2 membrane through AT1R activation, with the JAK2 pathway playing a central role. Further, inhibiting JAK2 reverses this effect, indicating its potential in hypertension treatment. This study provides novel insights into the role of Ang II in renal potassium excretion and hypertension pathophysiology.

Small Molecule NS11021 Promotes BK Channel Activation by Increasing Inner Pore Hydration.

The Ca2+ and voltage-gated big potassium (BK) channels are implicated in various diseases, including heart disease, asthma, epilepsy, and cancer, but remain an elusive drug target. A class of negatively charged activators (NCAs) have been demonstrated to promote the activation of several potassium channels including BK channels by binding to the hydrophobic inner pore, yet the underlying molecular mechanism of action remains poorly understood. In this work, we analyze the binding mode and potential activation mechanism of a specific NCA named NS11021 using atomistic simulations. The results show that NS11021 binding to the pore in deactivated BK channels is nonspecific and dynamic. The binding free energy of -8.3 ± 0.7 kcal/mol (KD = 0.3-3.1 μM) calculated using umbrella sampling agrees quantitatively with the experimental EC50 range of 0.4-2.1 μM. The bound NS11021 remains dynamic and is distal from the filter to significantly impact its conformation. Instead, NS11021 binding significantly enhances the pore hydration due to the charged tetrazol-phenyl group, thereby promoting the opening of the hydrophobic gate. We further show that the free energy barrier to K+ permeation is reduced by ∼3 kcal/mol regardless of the binding pose, which could explain the ∼62-fold increase in the intrinsic opening of BK channels measured experimentally. Taken together, these results support the idea that the molecular mechanism of NS11021 derives from increasing the hydration level of the conformationally closed pore, which does not depend on specific binding and likely explains the ability of NCAs to activate multiple K+ channels.

Effect of fibroblasts small- conductance Ca2+ -activated potassium channel subtype 2 (SK2) on myocardial fibrosis in pressure overload mouse

Studies have shown that Small conductance Ca2 + −activated K+ (SK) channel are expressed in fibroblasts. We aimed to determine the expression of SK2 channels in cardiac fibroblasts during myocardial hypertrophy and investigate its relationship with fibrotic remodeling. Myocardial hypertrophy and fibrotic remodeling induced by transverse aortic constriction (TAC) were assessed by echocardiography, Masson's trichrome staining and Western blot. Knockdown and overexpression of the SK2 protein were used to assess relationship between SK2 expression in fibroblasts and myocardial fibrosis. There is a positive correlation between myocardial fibrosis and SK2 channel protein expression during the development of myocardial hypertrophy. The differentiation and secretion of fibroblasts in mice with cardiac hypertrophy are enhanced, and the expression of SK2 channel protein is increased. Manipulating SK2 levels in fibroblasts can either promote or inhibit their differentiation and secretory function. Knocking down SK2 reduces the up-regulation of TGF β1, p-Smad2/3/GAPDH, p-p38/GAPDH, p-ERK1/2/GAPDH, and p-JNK/GAPDH proteins induced by Ang II in cardiac fibroblasts without significantly affecting total protein levels. AAV9-SK2-RNAi injection in mice improves cardiac function. Collectively, our study suggests that the expression of the SK2 channel is significantly increased in fibroblasts of mice with myocardial hypertrophy, potentially impacting myocardial fibrosis remodeling via the TGF-β signaling pathway.

The m6A methyltransferase METTL3 modifies Kcnk6 promoting on inflammation associated carcinogenesis is essential for colon homeostasis and defense system through histone lactylation dependent YTHDF2 binding

Inflammation induces tumor formation and plays a crucial role in tumor progression and prognosis. KCNK6, by regulating K(+) efflux to reduce NLRP3 Inflammasome-induced lung injury, relaxes the aorta. This study aims to elucidate the effects and biological mechanism of KCNK6 in inflammation-associated carcinogenesis, which may be essential for colon homeostasis and the defense system. To induce colitis, mice were given 3.0% Dextran Sodium Sulfate (DSS) in their drinking water for 7 days. The Azoxymethane (AOM) +DSS method was used to induce colon cancer in the mice model. Bone marrow-derived macrophages (BMDM) from Kcnk6-/- mice, AW264.7 cells, and human colon cancer HCT116 and Caco2 cells were used as in vitro models. The loss of Kcnk6 prevented spontaneous colitis and restored mucosal integrity and homeostatic molecules. Additionally, the loss of Kcnk6 reduced the severity of AOM/DSS-induced carcinogenesis. Kcnk6 promoted cell viability and proliferation in HCT-116 or Caco-2 cells. The loss of Kcnk6 inhibited the levels of inflammatory factors in BMDM cells. Kcnk6 accelerated potassium channel activity, inducing NLRP3 inflammasome activation. METTL3-mediated m6A modification increased Kcnk6 stability in a YTHDF2-dependent manner. Histone lactylation activated the transcription of YTHDF2/Kcnk6. Our study revealed the important role of Kcnk6 in inflammation-associated carcinogenesis progression. The m6A methyltransferase METTL3 and histone lactylation increased Kcnk6 stability in a YTHDF2-dependent manner, providing a potential strategy for inflammation-associated carcinogenesis or colorectal cancer therapy.

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.

Cantharidin and sodium fluoride attenuate the negative inotropic effect of the A1-adenosine receptor agonist N6-(R)-phenylisopropyl adenosine in isolated human atria.

Cantharidin and sodium fluoride inhibit the activity of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) and increase the force of contraction in human atrial preparations. R-phenylisopropyl adenosine (R-PIA) acts as an agonist at A1-adenosine receptors. R-PIA exerts a negative inotropic effect on human atria. The effect of R-PIA-and its various manifestations-are currently explained as a function of the inhibition of sarcolemmal adenylyl cyclase activity and/or opening of sarcolemmal potassium channels. We hypothesise that cantharidin and sodium fluoride may attenuate the negative inotropic effect of R-PIA. During open heart surgery, trabeculae carneae from the right atrium were obtained for human atrial preparations (HAPs). These trabeculae were mounted in organ baths and electrically stimulated at 1Hz. Furthermore, we studied isolated electrically stimulated left atrial (LA) preparations from female wild-type mice (CD1). The force of contraction was recorded under isometric conditions. R-PIA (1µM) exerted a rapid negative inotropic effect in the HAPs and mice LA preparations. These negative inotropic effects of R-PIA were attenuated by pre-incubation for 30min with 100-µM cantharidin in HAPs, but not in mice LA preparations. Adenosine signals via A1 receptors in a species-specific pathway in mammalian atria. We postulate that R-PIA, at least in part, exerts a negative inotropic effect via activation of serine/threonine phosphatases in the human atrium.

Straight from the heart – infrared light regulation of mitochondrial large conductance Ca2+-activated potassium channel activity in cardiomyocytes of Guinea Pig

Straight from the heart – infrared light regulation of mitochondrial large conductance Ca2+-activated potassium channel activity in cardiomyocytes of Guinea Pig

3-methoxycatechol causes vasodilation likely via KV channels: ex vivo, in silico docking and in vivo study

Substituted catechols include both natural and synthetic compounds found in the environment and foods. Some of them are flavonoid metabolites formed by the gut microbiota which are absorbed afterwards. Our previous findings showed that one of these metabolites, 4-methylcatechol, exerts potent vasorelaxant effects in rats. In the current study, we aimed at testing of its 22 structural congeners in order to find the most potent structure and to investigate the mechanism of action. 3-methoxycatechol (3-MOC), 4-ethylcatechol, 3,5-dichlorocatechol, 4-tert-butylcatechol, 4,5-dichlorocatechol, 3-fluorocatechol, 3-isopropylcatechol, 3-methylcatechol and the parent 4-methylcatechol exhibited high vasodilatory activities on isolated rat aortic rings with EC50s ranging from ∼10 to 24 μM. Some significant sex-differences were found. The most potent compound, 3-MOC, relaxed also resistant mesenteric artery but not porcine coronary artery, and decreased arterial blood pressure in both male and female spontaneously hypertensive rats in vivo without affecting heart rate. It potentiated the vasodilation mediated by cAMP and cGMP, but did not impact L-type Ca2+-channels. By using two inhibitors, activation of voltage-gated potassium channels (KV) was found to be involved in the mechanism of action. This was corroborated by docking analysis of 3-MOC with the KV7.4 channel. None of the most active catechols decreased the viability of the A-10 rat embryonic thoracic aorta smooth muscle cell line. Our findings showed that various catechols can relax vascular smooth muscles and hence could provide templates for developing new antihypertensive vasodilator agents without affecting coronary circulation.

Abstract Tu028: NADPH Oxidase Inhibition Antagonizes Endothelial Pro-inflammatory and Pro-oxidant Signaling Resulting in Enhanced Coronary Vasorelaxation in Diabetic Models

Introduction: NADPH oxidase (NOX) overactivation and reactive oxygen species amplification may underlie worse cardiac outcomes in patients with diabetes. Our group has shown impaired endothelium-dependent coronary vasodilation in diabetes, with restored endothelial function following NOX inhibition. Persistently high levels of oxidative stress may be driving this impaired coronary microvascular reactivity by altering small-conductance potassium channel activity. We hypothesized that NADPH oxidase inhibition would be vasoprotective through reductions in pro-oxidant and pro-inflammatory signaling. Methods: Human coronary arterial endothelial cells (HCAECs) were obtained from diabetic (D) and nondiabetic (C) patients (N = 4 per group). HCAECs were cultured in normo- or hyperglycemic conditions, with one hyperglycemic group receiving NADPH oxidase inhibitor apocynin (DT). Molecular signaling was assessed using immunoblotting. Results: Diabetes resulted in significantly increased pro-oxidant markers, including NOX 4 (p = 0.005), phosphorylated endothelial nitric oxide synthase (p-eNOS) (p = 0.007), and eNOS (p = 0.003). Pro-inflammatory Nrf2 was also increased (p = 0.0003), with a trend toward increased NFkB (p = 0.09) in D compared to C. Antioxidant peroxiredoxin (PRDX) 1 was significantly reduced in D compared to C (p = 0.02). NOX inhibition rescued these effects, with reversed trends in NOX 4 (p = 0.008), p-eNOS (p = 0.03), and PRDX 1 (p = 0.02) following apocynin treatment. NOX inhibition resulted in de novo decrease in NOX 1 in DT when compared to D (p = 0.008) and C (p = 0.02), and decrease in phosphorylated NFkB (p-NFkB) when compared to D (p = 0.02). There was a trend toward decreased total NFkB (p = 0.07) and p-NFkB:NFkB ratio (p = 0.06) and increased antioxidant thioredoxin (TXN) 2 (p = 0.05) in diabetic cells following treatment. Antioxidant superoxide dismutase (SOD) 2 (p = 0.01) and TXN 1 (p = 0.02) were increased in D when compared to C, while apocynin treatment reversed these changes (SOD 2, p = 0.0006; TXN 1, p = 0.04). There were no significant differences in NOX 2, SOD 1, catalase, PRDX 2, or PRDX 3 between the groups. Conclusions: Apocynin overall suppressed pro-oxidant and pro-inflammatory signaling in human diabetic coronary endothelial cells, resulting in improved coronary endothelium-dependent vasorelaxation. Cardioprotective strategies that incorporate NADPH oxidase inhibition in the setting of diabetes warrant further investigation.

Acute lipopolysaccharide (LPS)-induced cell membrane hyperpolarization is independent of voltage gated and calcium activated potassium channels

The gram-negative toxin lipopolysaccharides (LPS) are known to trigger inflammatory cytokines in mammals, which can result in pathological responses. Upon treatment of bacterial sepsis with antibiotics, the lysing bacteria can present a surge in LPS, inducing a cytokine storm. However, LPS can also have direct cellular effects, including transient rapid hyperpolarizing of the membrane potential, blocking glutamate receptors and even promoting release of glutamate. The detailed mechanism of action for these immediate responses is still unresolved. In addressing the membrane hyperpolarization, voltage gated K+ channel blockers 4-aminopyridine (4-AP, 3 mM), quinidine hydrochloride monohydrate (0.1 mM) and tetraethylammonium (TEA, 20 mM) were examined along with RNAi knockdowns of potential calcium activated K+ channels. The immediate responses of LPS were not blocked. Even in the presence of glutamate, the membrane still hyperpolarizes with LPS. When the driving gradient for the ionotropic glutamate receptors is enhanced during hyperpolarization, spontaneous quantal responses are dampened in amplitude. Thus, glutamate receptors are blocked, and the mechanism of hyperpolarization remains unresolved. The larval Drosophila glutamatergic neuromuscular junction is used as a model synaptic preparation to address the direct rapid actions by LPS.

Characterization of a novel Ca2+-Activated potassium channel in rat brain rough endoplasmic reticulum

ObjectivesPotassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). MethodsRats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to −60 mV following incorporation of rat brain RER vesicles into planar bilayers. ResultsWe observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. ConclusionOur study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.

GRT-X Stimulates Dorsal Root Ganglia Axonal Growth in Culture via TSPO and Kv7.2/3 Potassium Channel Activation.

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.

Update on Overactive Bladder Therapeutic Options.

Millions of Americans are burdened by overactive bladder (OAB) syndrome and the psychogenic and economic hardships that accompany it. Several theories attempt to explain OAB as a neurogenic dysfunction, myogenic dysfunction, urothelial dysfunction, or decreased expression of a channel protein secondary to bladder outlet obstruction. Given that the etiology of OAB is a working theory, the management of OAB is also an evolving subject matter in medicine. There are uncertainties surrounding the pathophysiology of OAB, the strength of a clinical diagnosis, and accurate reporting because of the disease's stigma and decreased use of health care. This is a narrative review that used PubMed, Google Scholar, Medline, and ScienceDirect to review literature on current and future OAB therapies. Currently, first-line treatment for OAB is behavioral therapy that uses lifestyle modifications, bladder-control techniques, and psychotherapy. Second-line therapy includes antimuscarinic agents or beta 3 adrenergic agonists, and studies have shown that combination therapy with antimuscarinics and beta 3 adrenergic agonists provides even greater efficacy than monotherapy. Third-line therapies discussed include onabotulinumtoxinA, posterior tibial nerve stimulation, and sacral neuromodulation. OnabotulinumtoxinA has been FDA-approved as a nonpharmaceutical treatment option for refractory OAB with minimal side effects restricted to the urinary tract. Posterior tibial nerve modulation and sacral neuromodulation are successful in treating refractory OAB, but the costs and complication rates make them high-risk procedures. Therefore, surgical intervention should be a last resort. Estrogen therapy is effective in alleviating urinary incontinence in postmenopausal women, consistent with the association between estrogen deficiency and genitourinary syndrome. Potassium channel activators, voltage-gated calcium channel blockers, and phosphodiesterase inhibitors look to be promising options for the future of OAB management. As new therapies are developed, individuals with OAB can better personalize their treatment to maximize their quality of life and cost-effective care.