Search Results (156)

The mineralocorticoid receptor (MR), traditionally associated with renal function, has also been identified in various extrarenal tissues, including the heart, brain, and dorsal root ganglion (DRG) neurons in rodents. Previous studies suggest a role for the MR in modulating peripheral nociception, with MR activation in rat DRG neurons by its endogenous ligand, aldosterone. This study aimed to determine whether MR, its protective enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), its endogenous ligand aldosterone, and the aldosterone-synthesizing enzyme CYP11B2 are expressed in human DRG neurons and whether they colocalize with key pain-associated signaling molecules as potential targets for genomic regulation. To this end, we performed mRNA transcript profiling and immunofluorescence confocal microscopy on human and rat DRG tissues. We detected mRNA transcripts for MR, 11β-HSD2, and CYP11B2 in human DRG, alongside transcripts for key thermosensitive and nociceptive markers such as TRPV1, the TTX-resistant sodium channel Nav1.8, and the neuropeptides CGRP and substance P (Tac1). Immunofluorescence analysis revealed substantial colocalization of MR with 11β-HSD2 and CGRP, a marker of unmyelinated C-fibers and thinly myelinated Aδ-fibers, in human DRG. MR immunoreactivity was primarily restricted to small- and medium-diameter neurons, with lower expression in large neurons (>70 µm). Similarly, aldosterone colocalized with CYP11B2 and MR with nociceptive markers including TRPV1, Nav1.8, and TrkA in human DRG. Importantly, functional studies demonstrated that prolonged intrathecal inhibition of aldosterone synthesis within rat DRG neurons, using an aldosterone synthase inhibitor significantly downregulated pain-associated molecules and led to sustained attenuation of inflammation-induced hyperalgesia. Together, these findings identify a conserved peripheral MR signaling axis in humans and highlight its potential as a novel target for pain modulation therapies. Full article

Patients with ulcerative colitis exhibit an increased risk for supraventricular arrhythmia during the active disease phase of the disease and show signs of atrial electrophysiological remodeling in remission. The goal of this study was to determine the basis for colitis-induced changes in atrial excitability. In a mouse model (C57BL/6; 3 months) of dextran sulfate sodium (DSS)-induced active colitis (3.5% weight/volume, 7 days), electrocardiograms (ECG) revealed altered atrial electrophysiological properties with a prolonged P-wave duration and PR interval. ECG changes coincided with a decreased atrial conduction velocity in Langendorff perfused hearts. Action potentials (AP) recorded from isolated atrial myocytes displayed an attenuated maximal upstroke velocity and amplitude during active colitis, as well as a prolonged AP duration (APD). Voltage clamp analysis revealed a colitis-induced shift in the voltage-dependent activation of the Na-current (I_Na) to more depolarizing voltages. In addition, protein levels of Na_v1.5 protein and connexin isoform Cx43 were reduced. APD prolongation depended on a reduction in the transient outward K-current (I_to) mostly generated by K_v4.2 channels. The changes in ECG, atrial conductance, and APD were reversible upon remission. The change in conduction velocity predominantly depended on the reversibility of the reduced Cx43 and Na_v1.5 expression. Treatment of mice with inhibitors of Angiotensin-converting enzyme (ACE) or Angiotensin II (AngII) receptor type 1 (AT1R) prevented the colitis-induced atrial electrophysiological remodeling. Our data support a colitis-induced increase in AngII signaling that promotes atrial electrophysiological remodeling and puts colitis patients at an increased risk for atrial arrhythmia. Full article

Neuropathic pain is a chronic and debilitating disorder of the somatosensory system that affects a significant proportion of the population and is characterized by abnormal responses such as hyperalgesia and allodynia. Voltage-gated ion channels, including sodium (Na_V), calcium (Ca_V), and potassium (K_V) channels, play a pivotal role in modulating neuronal excitability and pain signal transmission following nerve injury. This review intends to provide a comprehensive analysis of the molecular and cellular mechanisms by which dysregulation in the expression, localization, and function of specific Na_V channel subtypes (mainly Na_V1.7 and Na_V1.8) and their auxiliary subunits contributes to aberrant neuronal activation, the generation of ectopic discharges, and sensitization in neuropathic pain. Likewise, special emphasis is placed on the crucial role of Ca_V channels, particularly Ca_V2.2 and the auxiliary subunit Ca_Vα₂δ, whose overexpression increases calcium influx, neurotransmitter release, and neuronal hyperexcitability, thus maintaining persistent pain states. Furthermore, K_V channels (particularly K_V7 channels) function as brakes on neuronal excitability, and their dysregulation facilitates the development and maintenance of neuropathic pain. Therefore, targeting specific K_V channel subtypes to restore their function is also a promising therapeutic strategy for alleviating neuropathic pain symptoms. On the other hand, recent advances in the development of small molecules as selective modulators or inhibitors targeting voltage-gated ion channels are also discussed. These agents have improved efficacy and safety profiles in preclinical and clinical studies by attenuating pathophysiological channel activity and restoring neuronal function. This review seeks to contribute to guiding future research and drug development toward more effective mechanism-based treatments by discussing the molecular mechanisms underlying neuropathic pain and highlighting translational therapeutic opportunities. Full article

The insufficient selectivity of existing local anesthetics can lead to serious adverse effects. Considering the widespread use of this class of drugs, the development of new local anesthetics that do not cause side effects is an important task. One approach to address this issue is the use of photopharmacology, which enables the creation of agents with light-controlled biological activity. Several examples of azobenzene-based photoswitchable blockers of voltage-gated sodium (Na_v) channels have been described so far. These compounds can be used as light-controlled local anesthetics, one of which is ethercaine, synthesized by our group earlier. However, systematic studies of the “structure-activity” relationship in the series of light-controlled local anesthetics based on azobenzene are absent in the literature. The aim of this study was to obtain new derivatives of ethercaine and investigate their photophysical and biological properties. A total of 14 new derivatives were synthesized, and their structure was confirmed by various physicochemical analysis methods. The Z-E isomerization half-lifes were determined for all the synthesized compounds. The cytotoxic effect on normal cells was studied in vitro using human dermal fibroblasts (DF2). The local anesthetic activity of all the synthesized compounds was evaluated in vivo on a model of surface anesthesia in both darkness and under UV light irradiation. Based on the results obtained, conclusions were drawn regarding the potential of the proposed substances, and optimal pathways for structural modification were identified. Full article

Breast cancer remains the leading cause of cancer-related mortality among women worldwide, with limited therapeutic efficacy due to treatment resistance and adverse effects. Emerging evidence suggests that ion channels play crucial roles in tumor progression, regulating proliferation, apoptosis, migration, and metastasis. Voltage-gated potassium (Kv) and sodium (Nav) channels have been implicated in oncogenic signaling pathways. Scorpion venom peptides, known for their selective ion-channel-blocking properties, have demonstrated promising antineoplastic activity. This study explores the potential therapeutic applications of bioactive fractions derived from Chihuahuanus coahuilae, in breast cancer cell lines. Through chromatographic separation, mass spectrometry, and functional assays, we assess their effects on cell viability, proliferation, and ion channel modulation. Our preliminary data suggest that these venom-derived peptides interfere with cancer cell homeostasis by altering ion fluxes, promoting apoptosis, and inhibiting metastatic traits. These findings support the therapeutic potential of ion-channel-targeting peptides as selective anticancer agents. Further investigations into their molecular mechanisms may pave the way for novel, targeted therapies with improved efficacy and specificity for breast cancer treatment. Full article

Voltage-gated sodium (Nav) channels play a crucial role in initiating and propagating action potentials throughout the heart, muscles and nervous systems, making them targets for a number of drugs and toxins. While patch-clamp electrophysiology is considered the gold standard for measuring ion channel activity, its labor-intensive and time-consuming nature highlights the need for fast screening strategies to facilitate a preliminary selection of potential drugs or hazards. In this study, a high-throughput and cost-effective biosensing method was developed to rapidly identify specific agonists and inhibitors targeting the human Nav1.1 (hNav1.1) channel. It combines a red fluorescent dye sensitive to transmembrane potentials with CHO cells stably expressing the hNav1.1 α-subunit (hNav1.1-CHO). In the initial screening mode, the tested compounds were mixed with pre-equilibrated hNav1.1-CHO cells and dye to detect potential agonist effects via fluorescence enhancement. In cases where no fluorescence enhancement was observed, the addition of a known agonist veratridine allowed the indication of inhibitor candidates by fluorescence reduction, relative to the veratridine control without test compounds. Potential agonists or inhibitors identified in the initial screening were further evaluated by measuring concentration–response curves to determine EC₅₀/IC₅₀ values, providing semi-quantitative estimates of their binding strength to hNav1.1. This robust, high-throughput biosensing assay was validated through comparisons with the patch-clamp results and tested with 12 marine toxins, yielding consistent results. It holds promise as a low-cost, rapid, and long-term stable approach for drug discovery and non-target screening of neurotoxins. Full article

Voltage-gated sodium channels (Navs) are critical for membrane potential depolarisation in cells, with especially important roles in neuronal and cardiomyocyte membranes. Their malfunction results in a range of disorders, and they are the target of many widely used drugs. A rapid yet accurate functional assay is therefore desirable both to probe for novel active compounds and to better understand the many different Nav isoforms. Here, we use fluorescence to monitor Nav function: cells expressing either the cardiac Nav 1.5 or pain-associated Nav 1.7 were loaded with fluorescent membrane potential sensitive dye and then stimulated with veratridine. Cells expressing Nav 1.5 show a concentration-dependent slow rise and then a plateau in fluorescence. In contrast, cells expressing Nav 1.7 show a more rapid rise and then unexpected oscillatory behavior. Inhibition by flecainide and mexiletine demonstrates that these oscillations are Nav-dependent. Thus, we show that this fluorescent membrane potential dye can provide useful functional data and that we can readily distinguish between these two Nav isoforms because of the behavior of cells expressing them when activated by veratridine. We consider these distinct behaviors may be due to different interactions of veratridine with the different Nav isoforms, although more studies are needed to understand the mechanism underlying the oscillations. Full article

Scorpion venom peptides, particularly those derived from Asian species, have garnered significant attention, offering therapeutic potential in pain management, cancer, anticoagulation, and infectious diseases. This review provides a comprehensive analysis of scorpion venom peptides, focusing on their roles as voltage-gated sodium (Nav), potassium (Kv), and calcium (Cav) channel modulators. It analyzed Nav1.7 inhibition for analgesia, Kv1.3 blockade for anticancer activity, and membrane disruption for antimicrobial effects. While the low targeting specificity and high toxicity of some scorpion venom peptides pose challenges to their clinical application, recent research has made strides in overcoming these limitations. This review summarizes the latest progress in scorpion venom peptide research, discussing their mechanisms of action, therapeutic potential, and challenges in clinical translation. This work aims to provide new insights and directions for the development of novel therapeutic drugs. Full article

Fibroblast growth factor homologous factors (FHFs) regulate the activity of several different voltage-gated sodium channels (Na_vs). However, more work is needed to determine how specific FHF isoforms and variants affect the properties of different Na_v isoforms. In addition, it is not known if FHFs can differentially modulate the properties of Na_v variants associated with disease. Here, we investigated the effects of FHF2A and FHF2B on Nav1.1 properties as well as on a familial hemiplegic migraine 3 (FHM3) causing mutation in this channel, F1774S. We found that FHF2A, but not 2B, induced prominent long-term inactivation (LTI) in the wild-type (WT) Nav1.1. Interestingly, FHF2A induced LTI in the F1774S FHM3 mutant channel to a greater extent than in the WT. Furthermore, persistent currents caused by the F1774S mutation were attenuated by the co-expression of FHF2A, leading to a possible rescue of the mutant channel phenotype. By contrast, the P1894L mutation, which is associated with epilepsy and mild intellectual disability, greatly attenuated the LTI induced by FHF2A. Overall, our data show for the first time that FHF2A might be a significant modulator of Nav1.1 that can differentially modulate the impact of Nav1.1 disease-associated mutations. Full article

Several mutations of the uppermost arginine, R219, in the voltage-sensing sliding helix S4_I of cardiac sodium channel Nav1.5 are reported in the ClinVar databases, but the clinical significance of the respective variants is unknown (VUSs). AlphaFold 3 models predicted a significant downshift of S4_I in the R219C VUS. Analogous downshift S4_I, upon its in silico deactivation, resulted in a salt bridge between R219 and the uppermost glutamate, E161, in helix S2_I. To understand how salt bridge elimination affects biophysical characteristics, we generated mutant channel R219E, expressed it in the HEK293-T cells, and employed the patch-clamp method in a whole-cell configuration. Mutation R219E did not change the peak current density but shortened time to the peak current at several potentials, significantly enhanced activation, enhanced steady-state inactivation and steady-state fast inactivation, and slowed recovery from inactivation. Taken together, these data suggest that mutation R219E destabilized the resting state of Nav1.5. Cardiac syndromes associated with mutations R219P/H/C/P or E161Q/K are consistent with the observed changes of biophysical characteristics of mutant channel R219E suggesting pathogenicity of the respective VUSs, as well as ClinVar-reported VUSs involving arginine or glutamate in homologous positions of several Nav1.5 paralogs. Full article

The sigma-1 receptor (Sig-1R) has emerged as a significant target in the realm of pain management and has been the subject of extensive research. Nonetheless, its specific function in inflammatory pain within dorsal root ganglion (DRG) neurons remains inadequately elucidated. This study utilized whole-cell patch clamp techniques, single-cell real-time PCR, and immunohistochemistry to examine the influence of Sig-1R on inflammatory pain induced by complete Freund’s adjuvant (CFA) in a rat model. Our results revealed several key findings: (1) The expression of Sig-1R was found to be upregulated during the progression of inflammatory pain, with a notable translocation from the cytoplasm to the membrane; (2) Inhibition of peripheral Sig-1R using S1RA resulted in a reduction of CFA-induced allodynia; (3) Activation of Sig-1R through PRE-084 led to a decrease in the fast sodium current in isolated DRG neurons from CFA-treated rats, which was associated with a diminished action potential (AP) peak and maximum depolarizing rate (MDR), as well as an increased rheobase; (4) Furthermore, PRE-084 was observed to enhance the slow component of the sodium current, resulting in hyperpolarization of the threshold potential and an increase in AP firing frequency, alongside an elevation in the mRNA expression of the slow sodium channel Nav1.9 in CFA-treated rats. In conclusion, our findings suggest that the modulation of sodium channels by Sig-1R in DRG neurons plays a significant role in the mechanisms underlying inflammatory pain. Full article

Cenobamate is a new and highly effective antiseizure compound used for the treatment of adults with focal onset seizures and particularly for epilepsy resistant to other antiepileptic drugs. It acts on multiple targets, as it is a positive allosteric activator of γ-aminobutyric acid type A (GABA_A) receptors and an inhibitor of neuronal sodium channels, particularly of the late or persistent Na⁺ current. We recently evidenced the inhibitory effects of cenobamate on the peak and late current component of the human cardiac isoform hNav1.5. The determined apparent IC₅₀ values of 87.6 µM (peak) and 46.5 µM (late current) are within a clinically relevant range of concentrations (the maximal plasma therapeutic effective concentration for a daily dose of 400 mg in humans is 170 µM). In this study, we built a 3D model of the canonical hNav1.5 channel (UniProt Q14524-1) in open conformation using AlphaFold2, embedded it in a DPPC lipid bilayer, corrected the residue protonation state (pH 7.2) with H++, and added 2 Na⁺ ions in the selectivity filter. By molecular docking, we found the cenobamate binding site in the central cavity. We identified 10-point mutant variants in the binding site region and explored them via docking and MD. Mutants N1462K/Y (rs1064795922, rs199473614) and M1765R (rs752476527) (by docking) and N932S (rs2061582195) (by MD) featured higher predicted affinity than wild-type. Full article

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na⁺ (Na_V) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that Na_V channels, expressed in arteries, contribute to vascular tone in mouse mesenteric arteries (MAs). Here, we aimed to better characterize the mechanisms of action of VTD using mouse cecocolic arteries (CAs), a model of resistance artery. Using wire myography, we found that VTD induced vasorelaxation in mouse CAs. This VTD-induced relaxation was insensitive to prazosin, an α1-adrenergic receptor antagonist, but abolished by atropine, a muscarinic receptor antagonist. Indeed, VTD–vasorelaxant effect was totally inhibited by the Na_V channel blocker tetrodotoxin (0.3 µM), the NO synthase inhibitor L-NNA (20 µM), and low extracellular Na⁺ concentration (14.9 mM) and was partially blocked by the NCX1 antagonist SEA0400 (45.4% at 1 µM). Thus, we assumed that the VTD-induced vasorelaxation in CAs was due to acetylcholine release by parasympathetic neurons, which induced NO synthase activation mediated by the NCX1-Ca²⁺ entry mode in endothelial cells (ECs). We demonstrated NCX1 expression in ECs by RT-qPCR and immunohisto- and western immunolabelling. VTD did not induce an increase in intracellular Ca²⁺ ([Ca²⁺]i), while SEA0400 partially blocked acetylcholine-triggered [Ca²⁺]i elevations in Mile Sven 1 ECs. Altogether, these results illustrate that VTD activates Na_V channels in parasympathetic neurons and then vasorelaxation in resistance arteries, which could explain arterial hypotension after VTD intoxication. Full article

Dravet syndrome (DS) is a genetic disorder caused by a deficit in the Nav1.1 channel, leading to drug-resistant epilepsy. The Nav1.1 channel plays a crucial role in microglial cell activation, and microglia are recognized as key mediators of seizures. In this study, we explored the role of microglia in DS-related epileptogenesis using a knock-in mouse model (Scn1a^E1099X/+) that mimics a subset of DS patients. In these DS mice, we observed a significant downregulation of the Nav1.1 channel in microglia. This channel deficit led microglia to adopt a pro-inflammatory state in their quiescent phase. In the LPS-activated state, microglia predominantly exhibited an intermediate morphology rather than the expected fully activated form. The reduced expression of pro-inflammatory cytokines was detected in microglia following treatment with LPS. Notably, we found a significant decrease in the phagocytic ability of microglia in DS mice. Electrophysiological studies revealed an increased immature synaptic activity in the dentate gyrus in DS mice. The impaired microglial phagocytosis of damaged cells, combined with reduced cytokine secretion, may result in an excess of immature synaptic connections, neuronal hyperexcitation, and the formation of abnormal neural circuits in the hippocampus of Scn1a^E1099X/+ mice. These changes could potentially contribute to mechanisms relevant to epileptogenesis in DS. Full article

A possible molecular mechanism of the ligand–receptor binding of Ac-Lys-Lys-Lys-NH₂ (Ac-KKK-NH₂) to the Na_V1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH₂ application at 100 nM, a significant decrease in the effective charge carried by the Na_V1.8 channel activation gating system Z_eff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH₂ analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH₂ molecule upon binding to the Na_V1.8 channel are 11–12 Å, as revealed by the conformational analysis. The blind docking with the Na_V1.8 channel has made it possible to locate the Ac-KKK-NH₂ binding site on the extracellular side of the voltage-sensing domain VSD_I. The Ac-KKK-NH₂ amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Z_eff value. According to the results presented, Ac-KKK-NH₂ is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans. Full article