Search Results (215)

KCNV2 encodes Kv8.2, an electrically silent voltage-gated potassium channel subunit that is expressed in photoreceptors. Disease-causing variants in KCNV2 cause a monogenic disorder which is classified clinically as cone dystrophy with supernormal rod response (CDSRR). Here, we generated KCNV2-deficient human retinal organoids as a tool for gene therapy vector potency assessment. The organoids were derived from two separate sources: by generating IPSCs from patient blood and by gene editing of a control cell line. Eight KCNV2 gene therapy vectors were assessed in retinal organoids; Kv8.2 protein levels and its in situ interactions with potassium channel binding partners were quantitatively assessed. We show significant enhancements in vector potency and specificity by transgene codon optimisation and the use of the photoreceptor-specific rhodopsin kinase (RK) promoter, respectively. Single-cell RNA sequencing was performed in transduced retinal organoids to assess the performance of the AAV vectors at single-cell resolution. KCNV2-deficient photoreceptors had an upregulation in genes associated with apoptosis, oxidative stress, and hypoxia pathways which were partially restored in AAV-KCNV2 transduced photoreceptors. These data show how human retinal organoids can be used to evaluate AAV gene therapy vector potency in vitro in a physiologically relevant model for the selection of lead therapeutic candidates and to help minimise the use of animals in preclinical development. Full article

Background: Hypertension and its complications are a major global health problem, and natural compounds with vasorelaxant effects are being investigated as potential antihypertensive agents. Objective: This study aimed to determine whether rosmarinic acid (RA) induces vasorelaxation in the rat thoracic aorta and to elucidate the underlying mechanisms. Methods: Isolated thoracic aortic rings, with or without endothelium, were precontracted with phenylephrine and subsequently exposed to cumulative concentrations of RA. The roles of endothelium-derived factors, potassium channels, and calcium signaling were evaluated using selective pharmacological inhibitors and activators. In addition, the involvement of the AMPK pathway, adenylate cyclase/cAMP pathway, PKC signaling, β-adrenergic receptors, muscarinic receptors, and angiotensin II in RA-induced vasorelaxation was investigated. Results: RA induced a concentration-dependent vasorelaxation in endothelium-intact thoracic aortic rings (p < 0.001; pD₂ = 7.67 ± 0.04). The vasorelaxant effect of RA was attenuated in endothelium-denuded vessels (pD2: 5.26 ± 0.18). The relaxation response was significantly attenuated by inhibitors of the PI3K/Akt/eNOS/NO/cGMP pathway and by blockers of BK_Ca, IK_Ca, and Kv potassium channels (p < 0.001). Furthermore, RA markedly inhibited both extracellular Ca²⁺ influx and intracellular Ca²⁺ release from the sarcoplasmic reticulum (p < 0.001). RA incubation also significantly reduced the contractions induced by angiotensin II (Ang II) and by the PKC activator PMA (p < 0.001). Other tested pathways had no significant influence on the vasorelaxant effect of RA (p > 0.05). Conclusions: These findings demonstrate that rosmarinic acid induces both endothelium-dependent and endothelium-independent vasorelaxation in the rat thoracic aorta through activation of the PI3K/Akt/eNOS/NO/cGMP pathway, opening of BK_Ca, IK_Ca, and Kv potassium channels, and suppression of Ca²⁺ mobilization. Additionally, inhibition of PKC- and angiotensin II-mediated vascular contraction contributes to RA-induced vasorelaxation. RA may therefore have therapeutic potential in the management of hypertension. Full article

Calmodulin (CaM) plays a central role in cardiac excitation–contraction coupling by regulating ion channels, including the L-type calcium (Ca²⁺) channel Ca_v1.2 and the voltage-gated potassium (K⁺) channel K_v7.1. Mutations in CaM are linked to severe arrhythmogenic disorders such as Long QT syndrome (LQTS), yet the molecular mechanisms remain incompletely understood. Here, we investigate the structural and functional consequences of the arrhythmia-associated CaM variant D133H. Biophysical analysis revealed that D133H destabilises Ca²⁺ binding at the C-terminal lobe of CaM, altering its Ca²⁺-dependent conformational changes. Electrophysiological recordings demonstrated that CaM D133H impairs Ca²⁺-dependent inactivation (CDI) of Ca_v1.2, prolonging Ca²⁺ influx, while also reducing activation of K_v7.1, thereby limiting repolarising K⁺ currents. Together, these dual defects converge to prolong action potential duration, providing a mechanistic basis for arrhythmogenesis in LQTS. Our findings establish that CaM D133H perturbs both Ca²⁺ and K⁺ channel regulation, highlighting a shared pathway by which calmodulinopathy mutations disrupt cardiac excitability. Full article

Amyotrophic lateral sclerosis (ALS) is a multisystem progressive neurodegenerative disease. A recent theory of ALS onsetting pathogenesis proposed that the initiating primary damage is an acquired irreversible intrafusal proprioceptive terminal PIEZO2 channelopathy with underlying genetic and environmental risk factors. This Piezo2 channelopathy may also disrupt the ultrafast proton-based oscillatory signaling to motor neurons through vesicular transporter 1 (VGLUT1) and to the hippocampus through VGLUT2. As a result, it may gradually degenerate motor neurons in which process K_v1.2 ion channels are gradually depleted. It also gradually depletes heat shock transcription factor-1 (HSF-1) in the hippocampus, hence negatively affecting adult hippocampal neurogenesis. Syndecans, especially syndecan-3 (SDC3) in the nervous system, may act as critical players in the maintenance of the crosstalk between Piezo ion channels. Hence, our goal was to reanalyze the potential pathogenic gene variants from the cohort of our previous ALS study with a special focus on the aforementioned genes. Reanalysis of data formerly acquired by whole-exome sequencing of 21 non-related adult ALS patients was carried out with a focus on 28 genes. Accordingly, we identified charge-altering variants of SDC3 in 13 patients out of 21 that may contribute to the impairment of the Piezo crosstalk, and the progressive loss of the proposed proton-based signaling to motor neurons and to the hippocampus. A variant of uncertain significance was identified in the KCNA2 gene that may facilitate the faster loss of K_v1.2 ion function on motor neurons when Piezo2 channelopathy prevails. Not to mention that one variant was identified in the potassium current rectifying ion channels encoding KCNK1 and KCNK16 genes that may also propel the ALS disease process and provide the autoimmune-like pathogenic background. Moreover, Piezo2 channelopathy likely promotes diminishing HSF1 function in the hippocampus in the presence of the identified HSF1 variant. The current findings may support the ALS onsetting acquired irreversible Piezo2 channelopathy-induced pathogenesis. However, the preliminary nature of these findings needs validation and further functional studies on cohorts with a larger sample size in the future. Full article

Animal toxins contain various bioactive peptides and proteins which have evolved to interact in specific ways. As such, they are a good starting point for developing new drugs and vaccines. This paper examines three natural neurotoxins derived from the black mamba (Dendroaspis polylepis), which show significant pharmacological potential: mambalgins, fasciculins and dendrotoxins. All three may be of value in the treatment of pain, cancer and neurodegenerative disease. Mambalgins provide similar pain relief to opioids but without the risk of addiction; they act by selectively blocking acid-sensitive ion channels (ASICs), especially ASIC1a. Thanks to this inhibitory activity they also demonstrate selective activity against glioblastoma, melanoma and leukemia cells as innovative anticancer drugs. Fasciculins are very strong inhibitors of acetylcholinesterase (AChE) and hence offer promise in multi-target drugs and as treatments for treating Alzheimer’s disease. Dendrotoxins such as DTX-K and DTX-I are able to modulate neuronal excitability and synaptic transmission by blocking voltage-gated potassium channels (Kv1.1, Kv1.2, Kv1.6); both have been shown to be effective against cancer cells, and to influence the cardiovascular, immune, and digestive systems. Recent advances in recombinant biotechnology and protein engineering have allowed their safe production with increased therapeutic value. The review examines the translational potential of D. polylepis venom proteins and highlights the need for additional preclinical research on bioactive molecules of toxin origin. Full article

Mutation T226R in the Kv1.1 α-subunit of voltage-gated potassium Kv1 channels is associated with episodic ataxia type 1, severe neuromyotonia, and epilepsy. In vitro, this mutation was reported to considerably distort the functioning of homotetrameric channels Kv1.1; however, in the brain, Kv1.1 α-subunits form heterochannels predominantly associating with Kv1.2 α-subunits. Using the patch-clamp technique, fluorescent and Förster resonance energy transfer confocal microscopy, we revealed that heterochannels Kv(1.1(T226R)-1.2)₂ formed by concatemers Kv1.1(T226R)-Kv1.2 in Neuro-2a cells have significantly slower activation and deactivation rates, and their activation occurs at a much less negative membrane potential compared to channels Kv(1.1-1.2)₂ formed by concatemers Kv1.1-Kv1.2. This mutation does not noticeably affect the formation of complexes between α-subunits Kv1.1 and Kv1.2, but it does induce a delayed and possibly decreased presentation of heterochannels Kv(1.1(T226R)-1.2)₂ on the plasma membrane. At the same time, the T226R mutation has a much stronger negative effect on the membrane presentation of homotetrameric Kv1.1 channels. Since heterochannels Kv1.1-Kv1.2 but not homotetrameric channels Kv1.1 are present in the brain, the heterochannels bearing mutation T226R are most likely underlying the pathogenesis of the disease by decreasing the responsiveness of cells to mild membrane depolarization and, thus, increasing the excitability of neurons. Full article

Background/Objectives: Potassium (K⁺) channels are essential transmembrane proteins that regulate ion flow, playing a critical role in regulating action potentials and neuronal transmission. Although K⁺ channel openers (agonists, K⁺ Ag) are widely used in treating neurological and psychiatric disorders, their precise mechanisms of action remain unclear. Our study explored how K⁺ channel openers might influence the expression of voltage-gated K⁺ channels (Kv) in rat brain. Methods: Briefly, eight rats per group received intraperitoneal injections of diazoxide (Dia), chlorzoxazone (Chl), or flupirtine (Flu). Two hours post-injection, the prefrontal cortex (PFC), nucleus accumbens (NAc), dorsal striatum (dSTR), dorsal hippocampus (dHIP), and ventral hippocampus (vHIP) were collected for mRNA expression analysis of various Kv. Results: Dia administration altered expression of Kcna6 in the NAc, dSTR, and vHIP, and Kcnq2 in the PFC, dSTR, and dHIP. The mRNA levels of Kcna2 and Kcna3 changed in the NAc, dHIP, and vHIP, while Kcna6 expression increased in the PFC, dHIP, and vHIP of rats treated with Chl. Injection of Flu resulted in altered expression for Kcna1 in the NAc, dSTR, and dHIP; Kcna3 in the PFC, NAc, dHIP, and vHIP; Kcna6 in the dSTR, dHIP, and vHIP; and Kcnq2 and Kcnq3 in the PFC, dHIP, and vHIP. We also found dose-dependent changes. Conclusions: To our knowledge, this is the first study to identify the effects of potassium channel openers on gene expression within the mesocorticolimbic and nigrostriatal dopaminergic systems. These findings reveal a novel molecular mechanism underlying the action of these drugs in the brain. Importantly, our results have broader implications for translational neuroscience, particularly in the context of repurposing FDA-approved drugs, such as diazoxide and chlorzoxazone, for the treatment of neurological disorders. Full article

Ion channels regulate ion transport across cell or organelle membranes, playing an important role in various biological processes. Sodium channel Nav1.4 is critical to initiating and propagating action potentials in skeletal muscles, and its dysfunction is associated with a variety of diseases, such as non-dystrophic myotonias. In this study, U-actitoxin-Avd3i (Avd3i), a Kunitz-type toxin derived from Anemonia viridis, was expressed in prokaryotic systems and was subsequently purified via high-pressure liquid chromatography. Patch clamp recording showed that Avd3i inhibited Nav1.4 in a concentration-dependent manner, with an IC₅₀ of 25.43 μM. However, the toxin exerted no inhibitory activity in Nav1.5/Nav1.7 channels or Kv1.1/Kv1.3/Kv1.4/Kv4.2 potassium channels. Our study found that the sea anemone-derived toxin Avd3i inhibited sodium channel Nav1.4, providing a novel molecule that can act on the channel. Full article

Background/Objectives: The voltage-gated potassium channels of the Kv4 family (Kv4.1, Kv4.2, Kv4.3) regulate neuronal excitability and synaptic integration. The dysregulation of these channels has been linked to neurodegenerative diseases, such as Alzheimer’s disease (AD), spinocerebellar ataxias, amyotrophic lateral sclerosis (ALS), prion diseases, and Parkinson’s disease (PD). Current evidence is scattered across diverse models, and a systematic synthesis is lacking. This review seeks to compile and analyze data on Kv4 channel alterations in neurodegeneration, focusing on genetic variants, functional changes, and phenotypic consequences. Methods: A systematic search was conducted for peer-reviewed studies, including human participants, human-derived cell models, and relevant animal models. Studies were considered eligible if they investigated Kv4.1–Kv4.3 (encoded by gene encoding the Kv4.1-Kv4.3 α-subunit of voltage-gated A-type potassium channels (KCND1-KCND3)) expression, function, or genetic variants, as well as associated auxiliary subunits such as DPP6 (dipeptidyl peptidase–like protein 6) and KChIP2 (Kv channel–interacting protein 2), in neurodegenerative diseases. Both observational and experimental designs were considered. Data extraction included disease type, model, Kv4 subunit, functional or genetic findings, and key outcomes. Risk of bias was assessed in all included studies. Results: Kv4 channels exhibit significant functional and expression changes in various neurodegenerative diseases. In AD and prionopathies, reduced Kv4.1- and Kv4.2-mediated currents contribute to neuronal hyperexcitability. In spinocerebellar ataxias, KCND3 mutations cause loss- or gain-of-function phenotypes in Kv4.3, disrupting cerebellar signaling. In models of ALS and PD, Kv4 dysfunction correlates with altered neuronal excitability and can be modulated pharmacologically. Subunit modulators such as DPP6 and KChIP2 influence channel function and could represent therapeutic targets. Conclusions: Kv4 channels are crucial for neuronal excitability in multiple neurodegenerative contexts. Dysregulation through genetic or pathological mechanisms contributes to functional deficits, highlighting Kv4 channels as promising targets for interventions aimed at restoring electrical homeostasis and mitigating early neuronal dysfunction. Full article

Heterotetramerization of Kv1.1 and Kv1.2 α-subunits expands the functional diversity of voltage-gated potassium Kv1 channels in the central nervous system (CNS), thus necessitating the study of the properties of these heterochannels, including their interactions with ligands. We report on the expression, electrophysiological, and ligand-binding properties of human heterochannels Kv(1.1-1.2)₂ formed by dimeric concatemers Kv1.1-Kv1.2 fused with fluorescent protein mKate2 in Neuro-2a cells. Kv(1.1-1.2)₂ is a low-voltage-activated, highly active, non-inactivating channel with a fast activation rate. Its activation rate and half-maximum activation voltage are similar to that of the Kv1.1 channel, but differ from that of Kv1.2. This suggests that the membrane expression of Kv(1.1-1.2)₂ may functionally compensate for the absence of membrane presentation of homotetrameric Kv1.1 channels in CNS. Hongotoxin 1 fused with fluorescent protein GFP (HgTx-G) is shown to be a pore-blocking ligand of Kv(1.1-1.2)₂ with a dissociation constant of 100 pM. Using confocal microscopy and competitive binding assay, HgTx-G and cells expressing Kv(1.1-1.2)₂, the apparent dissociation constants of the complexes between Kv(1.1-1.2)₂ and peptides Ce1, Ce4, hongotoxin 1, MeKTx11-1, agitoxin 2, charybdotoxin, and scyllatoxin were evaluated to be 14, 33, 40, 250, 800, and >>3300 pM, respectively. Heterotetramerization of α-subunits has a different effect on the affinity of ligands compared to those for Kv1.1 and Kv1.2 channels. Full article

Contactin-associated protein-like 2 (CASPR2) is a transmembrane protein of the neurexin superfamily, essential for clustering voltage-gated potassium channels, particularly Kv1, at the juxtaparanodal regions of myelinated axons. This precise localisation is essential for maintaining normal axonal excitability and preventing aberrant signal propagation. Autoantibodies targeting CASPR2 have been associated with various neurological syndromes, notably peripheral nerve hyperexcitability (PNH), which presents clinically with neuromyotonia and myokymia. PNH is characterised by distinctive electrophysiological findings, including neuromyotonic discharges, myokymic discharges, and afterdischarges, which provide diagnostic value and insight into underlying pathophysiology. This review explores the mechanisms of anti-CASPR2-associated PNH, focusing on how antibody-mediated disruption of Kv1 channel clustering leads to altered axonal excitability. Current evidence suggests that both the distal and proximal segments of the axon are sites of pathological activity, where impairments in action potential termination and re-entry prevention result in spontaneous, repetitive discharges. While afterdischarges likely originate within the axon, the precise location—whether in the alpha-motoneuron soma or axon—is uncertain. The involvement of spinal inhibitory circuits has also been proposed, though it remains speculative. Understanding the neurophysiological features of anti-CASPR2-associated PNH is essential for improving diagnostic accuracy and guiding treatment strategies. Further research is needed to clarify the mechanisms of CASPR2-related hyperexcitability. Full article

Cancer has recently been proposed as a type of channelopathy due to the aberrant expression of various ion channels. Voltage-gated potassium (K⁺) channels (VGKCs) are notably upregulated during tumor proliferation, while voltage-gated sodium (Na⁺) channels are predominantly associated with the invasive stage of cancer progression. Among these, the Kv10.1 channel has been found to be overexpressed in breast cancer, making it a promising therapeutic target. 4-Aminopyridine (4-AP), a non-selective voltage-gated potassium channel blocker, has emerged as a potential novel agent for breast cancer treatment. In this study, we aimed to elucidate the mechanism of action of 4-aminopyridine in breast cancer cells. To investigate the involvement of various cell death pathways, cycloheximide (CHX) (a paraptosis inhibitor), Z-VAD-FMK (a pan-caspase inhibitor), and 2-Aminoethoxydiphenyl borate (2-APB) (a phosphoinositide 3-kinase [PI3K] inhibitor) were employed. Experiments were conducted using the MCF-7 human breast cancer cell line and the L929 mouse fibroblast cell line as a healthy control. Assessments included cell viability assays, intracellular calcium (Ca²⁺) and K⁺ concentration measurements, and plasma membrane potential analysis. Our findings aim to contribute to the understanding of the therapeutic potential and cellular effects of VGKC blockers, particularly 4-aminopyridine, in breast cancer treatment strategies. Full article

Acute Respiratory Distress Syndrome (ARDS) is a complex and devastating form of respiratory failure, with high mortality rates, for which there is no pharmacological treatment. The acute exudative phase of ARDS is characterized by severe damage to the alveolar–capillary barrier, infiltration of protein-rich fluid into the lungs, neutrophil recruitment, and high levels of inflammatory mediators. Rapid resolution of this reversible acute phase, with efficient restoration of alveolar functional integrity, is essential before the establishment of irreversible fibrosis and respiratory failure. Several lines of in vitro and in vivo evidence support the involvement of potassium (K⁺) channels—particularly KvLQT1, expressed in alveolar cells—in key cellular mechanisms for ARDS resolution, by promoting alveolar fluid clearance and epithelial repair processes. The aim of our study was to investigate whether pharmacological activation of KvLQT1 channels could elicit beneficial effects on ARDS parameters in an animal model of acute lung injury. We used the well-established bleomycin model, which mimics (at day 7) the key features of the exudative phase of ARDS. Our data demonstrate that treatments with the KvLQT1 activator R-L3, delivered to the lungs, failed to improve endothelial permeability and lung edema in bleomycin mice. However, KvLQT1 activation significantly reduced neutrophil recruitment and tended to decrease levels of pro-inflammatory cytokines/chemokines in bronchoalveolar lavages after bleomycin administration. Importantly, R-L3 treatment was associated with significantly lower injury scores, higher levels of alveolar type I (HTI-56, AQP5) and II (pro-SPC) cell markers, and improved alveolar epithelial repair capacity in the presence of bleomycin. Together, these results suggest that the KvLQT1 K⁺ channel may be a potential target for the resolution of the acute phase of ARDS. Full article

Ant venoms are rich sources of bioactive molecules, including peptide toxins with potent and selective activity on ion channels, which makes them valuable for pharmacological research and therapeutic development. Voltage-dependent potassium (Kv) channels, critical for regulating cellular excitability or cell cycle progression control, are targeted by a diverse array of venom-derived peptides. This study focuses on MYRTX_A4-Tb11a, a peptide from Tetramorium bicarinatum venom, which was previously shown to have a strong paralytic effect on dipteran species without cytotoxicity on insect cells. In the present study, we show that Tb11a exhibited no or low cytotoxicity toward mammalian cells either, even at high concentrations, while electrophysiological studies revealed a blockade of hKv1.3 activity. Additionally, Ta11a, an analog of Tb11a from the ant Tetramorium africanum, demonstrated similar Kv1.3 inhibitory properties. Structural analysis supports that the peptide acts on Kv1.3 channels through the functional dyad Y21-K25 and that the disulfide bridge is essential for biological activity, as reduction seems to disrupt the peptide conformation and impair the dyad. These findings highlight the importance of three-dimensional structure in channel modulation and establish Tb11a and Ta11a as promising Kv1.3 inhibitors. Future research should investigate their selectivity across additional ion channels and employ structure-function studies to further enhance their pharmacological potential. Full article

Voltage-gated potassium channels Kv7.1, encoded by the gene KCNQ1, play critical roles in various physiological processes. In cardiomyocytes, the complex Kv7.1-KCNE1 mediates the slow component of the delayed rectifier potassium current that is essential for the action potential repolarization. Over 1000 KCNQ1 missense variants, many of which are associated with long QT syndrome, are reported in ClinVar and other databases. However, over 600 variants are of uncertain clinical significance (VUS), have conflicting interpretations of pathogenicity, or lack germline information. Computational prediction of the damaging potential of such variants is important for the diagnostics and treatment of cardiac disease. Here, we collected 1750 benign and pathogenic missense variants of Kv channels from databases ClinVar, Humsavar, and Ensembl Variation and tested 26 bioinformatics tools in their ability to identify known pathogenic or likely pathogenic (P/LP) variants. The best-performing tool, AlphaMissense, predicted the pathogenicity of 195 VUSs in Kv7.1. Among these, 79 variants of 66 wildtype residues (WTRs) are also reported as P/LP variants in sequentially matching positions of at least one hKv7.1 paralogue. In available cryoEM structures of Kv7.1 with activated and deactivated voltage-sensing domains, 52 WTRs form intersegmental contacts with WTRs of ClinVar-listed variants, including 21 WTRs with P/LP variants. ClinPred and paralogue annotation methods consistently predicted that 21 WTRs of KCNE1 have 34 VUSs with damaging potential. Among these, 8 WTRs are contacting 23 Kv7.1 WTRs with 13 ClinVar-listed variants in the AlphaFold3 model. Analysis of intersegmental contacts in CryoEM and AlphaFold3 structures suggests atomic mechanisms of dysfunction for some VUSs. Full article