Search Results (702)

Grape pomace extract (GPE) from Vitis vinifera L. cv. Aglianico is rich in polyphenols with recognized cardioprotective properties, yet its direct electrophysiological effects on spontaneous cardiac activity have not been previously investigated. Here, we examined the chronotropic effects of GPE using two complementary models: HL-1 cardiomyocytes, assessed by whole-cell patch-clamp and intracellular Ca²⁺ imaging, and the Drosophila melanogaster larval heart tube, evaluated by optical recording. In HL-1 cells, chronic treatment with 25 µg/mL GPE for 48 h significantly reduced potential spontaneous action frequency and selectively prolonged the diastolic depolarization phase without altering action potential morphology, depolarization-activated currents, or cytosolic Ca²⁺ homeostasis. GPE reduced the hyperpolarization-activated funny current (I_f) density without shifting its voltage dependence. GPE-treated cells retained cAMP sensitivity, as both isoproterenol and intracellular 8-Br-cAMP significantly increased I_f amplitude, while ELISA quantification confirmed that global cAMP levels were unaffected by GPE. In Drosophila larvae, a cAMP-independent myogenic preparation, GPE administered in the diet significantly reduced heart rate. These findings demonstrate that Aglianico GPE exerts a negative chronotropic effect through a mechanism that reduces functional I_f density without altering cAMP availability or HCN channel voltage dependence, and reveal a cAMP-independent component of action conserved across phylogenetically distant species. Full article

Human endometrial mesenchymal stem/stromal cells (eMSCs) are widely used in laboratories and clinical applications to study various aspects of tissue engineering and regenerative medicine. Three-dimensional (3D) cultivated MSCs have a higher therapeutic efficacy compared to 2D culture. Ion channels are involved in maintaining many physiological cell functions, including proliferation, differentiation, apoptosis, and migration. This study describes the functional expression of voltage-gated sodium channels (NaV) in eMSCs and the role of these channels in cell migration. Using RT-PCR analysis and immunofluorescent microscopy, we identified the expression of almost all pore-forming alpha (NaV 1.1, 1.2, 1.4–1.9) and channel-modulating beta-NaV subunits (except beta2) in eMSCs. In the whole-cell patch-clamp configuration, channels activated by membrane depolarization of eMSC were detected. The channels were blocked by the selective NaV antagonist TTX in nanomolar concentrations. The NaV agonist veratridine at a concentration of less than 40 μM inhibited voltage-gated sodium currents, while 100 μM and above prevented channel inactivation. The wound healing assay showed that both TTX (10 μM) and veratridine (100 μM) reduced the migration properties (the wound healing rate) of eMSCs cultivated in 2D conditions compared to the control. An opposite effect by both agents was shown on the motility of eMSCs cultivated in 3D conditions, increasing the cell spreading rate from spheroids. Our data suggest that NaV channels are expressed in human eMSCs and play an important role in the regulation of stem cell migration; this regulatory mechanism significantly depends on the culture conditions of MSCs. Full article

Dravet syndrome (DS) is a severe, catastrophic childhood epilepsy predominantly caused by loss-of-function mutations in the SCN1A gene, which encodes the voltage-gated sodium channel Na_v1.1. In this study, we evaluated the therapeutic potential of 5-(Benzofuran-2-yl)-3-(2-chloro-4-fluorobenzyl)-1,3,4-oxadiazol-2(3H)-one (GM-90663), a novel small molecule designed to address the complex pathophysiology of DS. Using scn1lab knockout (KO) zebrafish larvae—a robust vertebrate model for DS—we demonstrated that GM-90663 significantly alleviates seizure-like behavioral movements and rescues deficit in cognitive-like functions. Whole-cell patch-clamp recordings in hippocampal slices revealed that GM-90663 modulates voltage-gated Na⁺ channel kinetics; specifically, it suppresses slow ramp-induced currents, thereby effectively attenuating neuronal hyperexcitability. Furthermore, neurochemical profiling indicated that GM-90663 treatment leads to a marked increase in endogenous serotonin (5-HT) levels in both wild-type and KO larvae. Molecular docking simulations and subsequent in vitro enzymatic assays confirmed that this elevation in serotonin is mediated through the potent inhibition of monoamine oxidase (MAO) activity. Collectively, our findings suggest that GM-90663 exerts its anti-seizure effects through a synergistic dual mechanism—stabilizing sodium channel conductance and elevating serotonergic activity—positioning it as a promising multi-target candidate for the treatment of DS. Full article

Background: Genistein and resveratrol are bioactive compounds isolated from plants, recognized for their diverse biological activities including anti-cancer properties. Both compounds are also known as modulators of various types of ion channels, including voltage-gated potassium channels, Kv1.3. These channels are widely expressed in normal and cancer tissues. Their activity is crucial in regulating cell proliferation and apoptosis in cells that express Kv1.3 channels. The potential clinical application of channel inhibitors may extend to treating cancers characterized by an overexpression of these channels. Methods: This study investigates the inhibitory effects of genistein and resveratrol on Kv1.3 channels expressed in the cancer cell line Jurkat T by applying a whole-cell patch clamp. Results: Applying both compounds at concentrations ranging from 3 μM to 90 μM leads to a dose-dependent inhibition of channel activity, reducing it to approximately 50% of the control level. This inhibitory effect was reversible and associated with a significant reduction in the activation rate. When combined with simvastatin, the inhibitory effect exhibited synergy; however, it was additive when co-applied with mevastatin. Conclusions: The channel inhibition may putatively be linked to the anti-cancer activities of these compounds on Kv1.3 channel-expressing cancer cells, especially when co-applied with the statins. Full article

Background/Objectives: Copper (Cu²⁺) is an essential trace element that participates as a cofactor in key metabolic enzymes such as cytochrome c oxidase and superoxide dismutase. However, excessive copper exposure can be toxic and disturbances in copper homeostasis have been associated with neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Despite growing evidence linking copper to neuronal dysfunction, the cellular mechanisms by which Cu²⁺ affects neuronal excitability and neurotransmission remain poorly understood. The aim of this study was to investigate the effects of acute Cu²⁺ exposure on ionic currents involved in cellular excitability and neurotransmitter release in bovine chromaffin cells. Methods: Primary cultures of bovine chromaffin cells were used as a neuroendocrine model to study cellular excitability. Voltage-dependent ionic currents were recorded using the whole-cell patch-clamp technique in voltage-clamp configuration. Catecholamine secretion was monitored by amperometry, and cytosolic Ca²⁺ dynamics were measured in fluo-4-loaded cells during depolarization induced by high K⁺ stimulation. Results: Acute Cu²⁺ exposure produced a concentration-dependent enhancement of depolarization-evoked catecholamine release. In parallel, Cu²⁺ inhibited voltage-dependent calcium (ICa), sodium (INa), potassium (IKv), and calcium/voltage-dependent potassium (IKCa-v) currents in a concentration-dependent and partially reversible manner. In addition, Cu²⁺ increased basal cytosolic Ca²⁺ levels while reducing the amplitude of depolarization-evoked Ca²⁺ transients. Conclusions: Acute Cu²⁺ exposure exerts a dual effect in bovine chromaffin cells, inhibiting the ionic currents that support cellular excitability while potentiating catecholamine secretion. This apparent paradox is consistent with a disruption of intracellular Ca²⁺ homeostasis, in which elevated basal cytosolic Ca²⁺ may facilitate exocytosis despite reduced depolarization-evoked Ca²⁺ entry. These findings provide new insight into the mechanisms by which copper may alter neuronal signaling and contribute to neurotoxicity. Full article

Background: Corylin (3-(2,2-dimethylchromen-6-yl)-7-hydroxychromen-4-one), a bioactive flavonoid, has been reported to exercise anti-inflammatory, antineoplastic, and antioxidant effects, and may also possess lifespan-extending properties. Objectives: Any modifications of transmembrane ionic currents produced by corylin remain largely unknown. Methods: The patch-clamp technique and docking prediction were used in this study. Results: In pituitary GH₃ somatolactotrophs, corylin concentration-dependently increased the magnitude of the M-type K⁺ current (I_K(M)), with an EC₅₀ of 3.8 μM. Concurrently, the activation time constant of I_K(M) was shortened. The addition of linopirdine (10 μM), an I_K(M) inhibitor, suppressed the current amplitude. Corylin also induced a leftward shift in the steady-state activation curve and enhanced I_K(M) during pulse-train stimulation. Moreover, corylin increases the hysteretic strength of I_K(M) evoked by a long-lasting triangular ramp pulse; this effect was attenuated by linopirdine. The stimulatory effect of corylin on I_K(M) was not altered by carvedilol or iberiotoxin but was reduced by dapagliflozin. In contrast, depolarization-activated I_K(M) was not affected by 17β-estradiol alone. In cell-attached recordings, corylin increased M-type K⁺ (K_M)-channel activity with minimal change in single-channel amplitude, while prolonging the mean open time. This stimulatory effect was reversed by linopirdine or dapagliflozin. Additionally, corylin slightly inhibited the erg-mediated current. Docking analysis further suggested that corylin potentially interacts with residues in KCNQ2 or KCNH2 channels via hydrogen bonding and hydrophobic interactions. Conclusions: These findings suggest that corylin modulates ionic currents, primarily through K_M (KCNQ/K_V7) channels, which may underlie its in vivo actions and those of related flavonoids. These effects may contribute to the regulation of functional activities of neuronal, neuroendocrine, and endocrine cells. Full article

Saxitoxin (STX) is one of the most potent natural neurotoxins known and is the only marine toxin to be declared a chemical weapon. In both marine and freshwater systems filter feeding organisms can accumulate saxitoxin and human consumption of toxin-contaminated food can result in paralytic shellfish poisoning. Here we highlight for the first time a human cell-based assay for the detection and neutralisation of STX activity on an automated patch clamp (APC) system. We demonstrate that a human embryonic kidney (HEK) cell line expressing human Nav1.6 can rapidly and sensitively detect the presence of a range of sodium ion channel blockers including STX. The use of neutralising monoclonal antibody GT13-A and/or saxiphilin was found to confer specificity to the assay by being able to dissociate between STX (along with closely related analogues) and tetrodotoxin. Finally, the application of the functional assay for the detection of STX in complex samples was evaluated during an international exercise led by the Organisation for the Prohibition of Chemical Weapons (OPCW). The neutralisation of STX activity in blinded samples enabled the indirect detection of the toxin in the relevant samples and provided an alternative orthogonal technique to corroborate the findings of liquid chromatography–mass spectrometry (LC-MS). Collectively this work demonstrates the significant potential for functional assays in the analysis of samples suspected of being contaminated with STX and related sodium ion channel targeting toxins; complementing traditional direct identification methods such as high-performance liquid chromatography with fluorescence detection (HPLC-FLD), LC-MS or enzyme-linked immunosorbent assay (ELISA). Full article

The human corneal endothelium (HCE) is critical for maintaining corneal transparency. Dysfunctions due to cell loss are linked to altered intracellular calcium ([Ca²⁺]_i) homeostasis. Transient receptor potential channels (TRPs) are key regulators of [Ca²⁺]_i, and both L-ascorbic acid (Asc) and cannabinoid receptor (CB) agonists have been implicated in modulating TRP activity. This study investigated the effects of 1 mM Asc and the CB agonist WIN 55,212-2 (WIN) (10 µM) on [Ca²⁺]_i regulation in human corneal endothelial cells (HCECs). HCEC-12 was used as the established HCE cell model. [Ca²⁺]_i dynamics were assessed by fura-2/AM fluorescence imaging, and membrane currents were analyzed using planar patch-clamp recordings. Adding 1 mM Asc increased [Ca²⁺]_i, which was partially suppressed by the TRPV1 blocker AMG-9810 (AMG) (20 µM) and the TRPV4 blocker GSK2193874 (GSK219) (10 µM). Furthermore, 1 mM Asc increased whole-cell currents. WIN also induced [Ca²⁺]_i transients that were partially attenuated by AMG, the TRPM8 blocker AMTB (20 µM), GSK219, and the CB1 inverse agonist AM251 (10 µM). In addition, combined treatment with Asc and WIN enhanced [Ca²⁺]_i elevations compared with either treatment alone. These findings provide the first evidence for a functional interaction between TRP channel activity and CB signaling in HCECs. The inhibitory effect of AM251 suggests a predominant contribution of CB1 receptors. Given the central role of Ca²⁺ homeostasis in corneal endothelial function and disease, these results may contribute to a better understanding of endothelial pathophysiology and support further investigation of TRPs and cannabinoid signaling as potential targets in corneal disorders. Full article

The hERG potassium channel is critical for cardiac ventricular repolarization and a core target in pre-clinical drug safety screening. A robust, stable cell line with uniform, high hERG expression is essential for high-throughput assessments. In this study, we established a functional stable HEK293T cell line with high hERG expression. The hERG gene was subcloned into Lenti-HA-hERG-P2A-EGFP plasmid, in which GFP serves as a selection marker via a P2A self-cleaving peptide. GFP-positive monoclonal cells were isolated by fluorescence-activated cell sorting (FACS). Confocal imaging confirmed that hERG localized predominantly to the cell membrane, consistent with its physiological role. Manual patch-clamp revealed canonical hERG current properties: a small, stable current during depolarization to 20 mV, followed by a large outward tail current upon repolarization to −40 mV-a hallmark of hERG channel gating. Automated patch-clamp (APC)-based current profiling showed 93.5% of stable hERG cells exhibited peak tail currents > 50 pA (87% > 100 pA, with 49.5% > 400 pA), whereas 100% of blank HEK293T cells showed peak tail currents < 50 pA. Pharmacological validation with E-4031 demonstrated concentration-dependent inhibition of hERG currents, with an IC₅₀ of 29.8 nM, which is consistent with literature-reported values. The stable hERG-expressing HEK293T cell line developed here exhibits consistent hERG expression, canonical channel function, and physiological sensitivity to hERG blockers. When paired with high-throughput APC systems, this cell model provides a robust, standardized platform for pre-clinical drug-induced hERG inhibition evaluation, aiding early detection of long QT syndrome risks and safer drug development. Full article

Background/Objectives: Patient-derived mesenchymal stem cells (MSCs) represent a promising avenue for myocardial regeneration, yet therapeutic application remains limited by inconsistent differentiation capacity and the absence of standardized cardiogenic induction protocols. This study demonstrates a proof-of-concept for guiding patient-specific bone marrow MSCs toward a functional cardiomyocyte phenotype using paracrine signals from differentiating iPSC-derived cardiomyocytes (iPSC-CMs). Materials and Methods: MSCs were maintained in conditioned medium from a concurrent, validated iPSC-CM differentiation protocol, with evaluation via immunocytochemistry, optical mapping, and whole-cell patch-clamp recordings. Results: Differentiated MSCs acquired organized sarcomeric architecture with cross-striations and displayed spontaneous calcium oscillations with decay kinetics matching source iPSC-CMs (CaT50 ≈ 283 ms vs. 301 ms). In co-culture, MSC-derived cells exhibited synchronized calcium dynamics with iPSC-CMs, confirming functional coupling, while patch-clamp detected hallmark cardiac ion currents (INa, ICa,L, and IKv). Morphologically, MSC-CMs displayed more mature, elongated rod-like shapes. Conclusions: Although current densities indicate partial immaturity, their reproducible detection validates successful cardiomyogenic commitment. This “parallel differentiation” platform eliminates donor-specific protocol tuning, providing a streamlined, paracrine-mediated approach to generate autologous cardiomyocyte-like cells for disease modeling, pharmacological testing, and future regenerative applications. Full article

Two-pore channels (TPCs) are evolutionarily conserved intracellular cation channels found in both plants and animals, where they mediate ion fluxes across endomembrane compartments. While historically the plant channel was among the first plant ion channels to be characterized, thanks to the relative ease of applying the patch-clamp technique to isolated plant vacuoles, where it is localized, the functional properties of the two main human isoforms, HsTPC1 and HsTPC2, expressed in endosomal and lysosomal membranes, were elucidated much later. In plants, TPCs are typically represented by a single isoform, exemplified by AtTPC1 in the model plant Arabidopsis thaliana, which functions as a voltage-dependent, Ca²⁺-regulated channel. The physiological role of plant TPCs is not yet fully clarified, although evidence suggests that they may contribute to systemic signaling and stress responses. In humans, two main isoforms, HsTPC1 and HsTPC2, are expressed in endosomal and lysosomal membranes. Human TPCs are primarily regulated by the phosphoinositide PI(3,5)P₂ and display a high selectivity for Na⁺. However, these channels also appear as a non-selective cationic conductance when activated by the potent Ca²⁺-mobilizing messenger NAADP, likely through interaction with an accessory protein. Functionally, human TPCs are involved in endolysosomal trafficking, membrane fusion, and intracellular signaling, with emerging roles in immunity, metabolism, and disease. Overall, TPCs represent key components of intracellular ion homeostasis and cellular physiology; however, their precise regulatory mechanisms and integrated physiological roles remain only partially understood and, in several respects, are still elusive. Full article

Background: Neonatal and infant aortic arch reconstruction remains a high-risk cardiovascular procedure requiring effective cerebral and myocardial protection. Variability in perfusion strategies may influence early hemodynamic stability and postoperative recovery. This study aimed to evaluate the early and short-term cardiovascular outcomes of a standardized beating-heart aortic arch reconstruction strategy incorporating simultaneous antegrade selective cerebral and continuous coronary perfusion. Methods: In this retrospective single-center cohort study, 31 consecutive neonates and infants undergoing aortic arch reconstruction between November 2022 and December 2025 were analyzed. A standardized surgical protocol was applied, consisting of extensive ductal tissue resection, interdigitating posterior end-to-end anastomosis, anterior autologous pericardial patch augmentation, and moderate hypothermic antegrade selective cerebral perfusion combined with continuous coronary perfusion via innominate artery cannulation. Early postoperative outcomes and short-term echocardiographic follow-up results were assessed. Results: The cohort included 31 patients, 22.6% of whom had complex associated cardiac anomalies requiring concomitant procedures. Median cardiopulmonary bypass and aortic cross-clamp times were 119 and 64 min, respectively. There was no in-hospital mortality. Major complications were infrequent, and median intensive care unit stay was 5 days. During a median follow-up of 6.8 months, one patient (3.2%) developed recoarctation requiring reintervention. No late mortality was observed. Conclusions: A fully standardized beating-heart aortic arch reconstruction strategy incorporating simultaneous cerebral and coronary perfusion demonstrated favorable early cardiovascular and short-term outcomes, even in anatomically complex cases. Preservation of continuous coronary perfusion may be associated with improved myocardial stability and early postoperative recovery; however, these findings should be interpreted as observational and hypothesis-generating given the absence of a control group. Larger multicenter studies with longer follow-up are warranted to confirm these findings. Full article

Skeletal muscle differentiation is tightly regulated by membrane potential dynamics and voltage-dependent ion channel activity. Potassium (K⁺) and calcium (Ca²⁺) currents cooperate to orchestrate the transition of myoblasts into fusion-competent myotubes, and alterations in this process are associated with dystrophic phenotypes. Here, we investigated the electrophysiological remodeling accompanying C2C12 myogenesis and the modulatory effects of the polyphenol resveratrol (RES) on calcium voltage-gated channel subunit alpha 1 S (CACNA1S, Cav1.1, L-type) currents. Whole-cell patch-clamp recordings were performed in proliferating and differentiating C2C12 cells to characterize the temporal expression of K⁺ currents and voltage-dependent Ca²⁺ channels (VDCCs). During differentiation, three electrophysiological subpopulations were identified according to K⁺ current profiles: SK4+/EAG−/Kir−, SK4−/EAG+/Kir−, and SK4−/EAG+/Kir+. This sequence paralleled a progressive membrane hyperpolarization from −20 mV to −70 mV, consistent with the physiological maturation of myogenic cells. In C2C12 myocytes, nimodipine-sensitive L-type currents were the only Ca²⁺ conductance observed. Their activation threshold (~−30 mV) and half-activation voltage (V/2 ≈ −12 mV) indicated the co-expression of embryonic and adult Cav1.1 isoforms. Exposure to RES (30 µM, 48 h) produced a depolarizing shift in activation (ΔV/2 ≈ +9 mV) and a reduction in current amplitude across all voltages, consistent with a transition toward the adult splice variant of Cav1.1. These findings suggest that RES promotes electrophysiological maturation of skeletal muscle cells by modulating calcium channel expression and gating behavior. Given its known ability to correct splicing abnormalities in CACNA1S and related genes, resveratrol emerges as a promising pharmacological agent for restoring calcium homeostasis in neuromuscular disorders such as myotonic dystrophy type 1 (DM1). Full article

Spreading depolarizations (SDs) are major pathophysiological events in several brain diseases, including migraine, brain ischemia, trauma, and epilepsy. However, the electrophysiological detection of SDs remains challenging. In this study, we examined changes in spikes (action potentials (APs) and action currents (ACs)) in layer 5 neurons of the somatosensory cortex of anesthetized rats during transient excitation at the onset of high-potassium-induced SDs. During whole-cell recordings, spike amplitude progressively decreased while spike duration increased during gradual neuronal depolarization at SD onset, culminating in depolarization block. A similar decrease in spike amplitude and increase in spike duration were observed during the pre-SD excitation phase in loose cell-attached recordings from single neurons and in cluster analysis of extracellular spikes. Multiple (non-clustered) unit activity also showed decrease in spike amplitude and spike broadening during pre-SD excitation. These findings suggest that dynamic changes in spike amplitude and duration at SD onset could serve as markers for SD detection. Full article

Neuron-targeted therapies for Alzheimer’s disease (AD) have shown limited efficacy, highlighting the need to explore glial-based mechanisms of neuroprotection. Here, we show that astrocyte mitochondrial uncoupling via viral overexpression of uncoupling protein 4 (UCP4) restores neuronal circuits and ion channel function in aged 3xTG AD mice with overt symptoms. Spontaneous local field potential recordings revealed a partial recovery of hippocampal and subicular sharp wave ripple oscillations, electrophysiological signatures of neuronal circuits known to be altered in AD. Combined whole-cell patch-clamp electrophysiology with two-photon Ca²⁺ imaging further demonstrated that UCP4 modulates activity-dependent Ca²⁺ influx, A-type potassium channel function, and enhances glial cell line-derived neurotrophic factor (GDNF) signaling. These findings identify astrocytic mitochondrial uncoupling as a potent mechanism enhancing neuronal resilience and restoring circuit function in symptomatic AD brains. Full article