Search Results (13)

Toxins as channel probes, small guanidinium alkaloids, such as tetrodotoxin and saxitoxin, canonical pore occlusion in voltage-gated Na⁺ channels. Cystine-rich peptides from spiders, scorpions, cone snails, and sea anemones, which act as pore blockers or gating modifiers targeting voltage-sensing domains. Recent structural and electrophysiological studies have identified specific binding sites on ion channels, including the S5–S6 pore loops, outer vestibule and turret regions, and S3–S4 “paddle” motifs in NaV, Kv, and CaV channels. These discrete binding epitopes are recognized by different peptide toxins, enabling isoform- and state-specific modulation; for example, μ-conotoxins bind the NaV pore, whereas charybdotoxin and agitoxin target the Kv outer vestibule. Beyond mechanistic insights, peptide toxins inspire translational strategies, including emerging therapies for retinal degenerative diseases. Photopharmacology using chemical photoswitches allows reversible, light-controlled modulation of ion channels in retinal ganglion cells without genetic manipulation or cell transplantation. Although BENAQ was discovered by small-molecule screening rather than toxin-guided design, its ion channel control demonstrates the potential of toxin-based molecular determinants for engineering synthetic compounds. This review thus integrates structural, functional, and translational perspectives, emphasizing the versatility of animal-derived peptide toxins as molecular probes and as blueprints for precision ion channel modulation in health and disease. Full article

Podocytes express large-conductance Ca²⁺-activated K⁺ channels (BK channels) and at least two different pore-forming KCa1.1 subunit C-terminal splice variants, known as VEDEC and EMVYR, along with auxiliary β and γ subunits. Podocyte KCa1.1 subunits interact directly with TRPC6 channels and BK channels become active in response to Ca²⁺ influx through TRPC6. Here, we confirmed that Ca²⁺ influx through TRPC channels is reduced following the blockade of BK channels by paxilline. The overall abundance of KCa1.1 subunits, as well as that of β4 and γ3 subunits, were increased in glomeruli isolated from Sprague Dawley rats during chronic puromycin aminonucleoside (PAN) nephrosis. Exposing cultured mouse podocytes for 24 h to recombinant TNFα, a circulating factor implicated in pediatric nephrotic syndromes, did not affect the total abundance of KCa1.1, but did evoke significant increases in both β4 and γ3. However, TNFα evoked a marked increase in the surface abundance of KCa1.1 subunits, similar to that of its previously reported effects on TRPC6 channels. The effect of TNFα on the surface expression of KCa1.1 was eliminated following siRNA knockdown of the β4 subunits, suggesting a role for this subunit in KCa1.1 trafficking to the cell surface. By contrast, treating podocytes with suPAR did not affect the total or surface expression of KCa1.1. The coordinated activation of KCa1.1 channels may promote Ca²⁺ influx through TRPC channels during normal and abnormal podocyte function by maintaining a membrane potential that allows for the efficient permeation of divalent cations through TRPC pores. Full article

Bacterial protein toxins are secreted by certain bacteria and are responsible for mild to severe diseases in humans and animals. They are among the most potent molecules known, which are active at very low concentrations. Bacterial protein toxins exhibit a wide diversity based on size, structure, and mode of action. Upon recognition of a cell surface receptor (protein, glycoprotein, and glycolipid), they are active either at the cell surface (signal transduction, membrane damage by pore formation, or hydrolysis of membrane compound(s)) or intracellularly. Various bacterial protein toxins have the ability to enter cells, most often using an endocytosis mechanism, and to deliver the effector domain into the cytosol, where it interacts with an intracellular target(s). According to the nature of the intracellular target(s) and type of modification, various cellular effects are induced (cell death, homeostasis modification, cytoskeleton alteration, blockade of exocytosis, etc.). The various modes of action of bacterial protein toxins are illustrated with representative examples. Insights in toxin evolution are discussed. Full article

After parturition, bovine endometrial epithelial cells (BEECs) undergo serious inflammation and imbalance between oxidation and antioxidation, which is widely acknowledged as a primary contributor to the development of endometritis in dairy cows. Nevertheless, the mechanism of oxidative stress-mediated inflammation and damage in bovine endometrial epithelial cells remains inadequately defined, particularly the molecular pathways associated with mitochondria-dependent apoptosis. Hence, the present study was designed to explore the mechanism responsible for mitochondrial dysfunction-induced BEEC damage. In vivo, the expressions of proapoptotic protein caspase 3 and cytochrome C were increased significantly in dairy uteri with endometritis. Similarly, the levels of proapoptotic protein caspase 3, BAX, and cytochrome C were markedly increased in H₂O₂-treated BEECs. Our findings revealed pronounced BEEC damage in dairy cows with endometritis, accompanied by heightened expression of cyto-C and caspase-3 both in vivo and in vitro. The reduction in apoptosis-related protein of BEECs due to oxidant injury was notably mitigated following N-acetyl-L-cysteine (NAC) treatment. Furthermore, mitochondrial vacuolation was significantly alleviated, and mitochondrial membrane potential returned to normal levels after the removal of ROS. Excessive ROS may be the main cause of mitochondrial dysfunction. Mitochondrial permeability transition pore (mPTP) blockade by cyclophilin D (CypD) knockdown with CSA significantly blocked the flow of cytochrome C (cyto-C) and Ca²⁺ to the cytoplasm from the mitochondria. Our results indicate that elevated ROS and persistent opening of the mPTP are the main causes of oxidative damage in BEECs. Collectively our results reveal a new mechanism involving ROS-mPTP signaling in oxidative damage to BEECs, which may be a potential avenue for the clinical treatment of bovine endometritis. Full article

Huntington’s disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca²⁺ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca²⁺ homeostasis. Lysosomes are intracellular Ca²⁺ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca²⁺ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca²⁺ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca²⁺ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca²⁺ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca²⁺ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy. Full article

Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells. Full article

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey’s excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K⁺-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4–5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads. Full article

Multiple myeloma is a malignant expansion of plasma cells and aggressively affects bone health. We show that P2X7 receptor altered myeloma growth, which affects primary bone cells in vitro. Expression on six human myeloma cell lines confirmed the heterogeneity associated with P2X7 receptor. Pharmacology with 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) as agonist showed dose-dependent membranal pores on RPMI-8226 (p = 0.0027) and blockade with P2X7 receptor antagonists. Ca²⁺ influx with increasing doses of BzATP (p = 0.0040) was also inhibited with antagonists. Chronic P2X7 receptor activation reduced RPMI-8226 viability (p = 0.0208). No apoptosis or RPMI-8226 death was observed by annexin V/propidium iodide (PI) labeling and caspase-3 cleavage, respectively. However, bromodeoxyuridine (BrdU) labelling showed an accumulation of RPMI-8226 in the S phase of cell cycle progression (61.5%, p = 0.0114) with significant decline in G0/G1 (5.2%, p = 0.0086) and G2/M (23.5%, p = 0.0015) phases. As myeloma pathology depends on a positive and proximal interaction with bone, we show that P2X7 receptor on RPMI-8226 inhibited the myeloma-induced suppression on mineralization (p = 0.0286) and reversed the excessive osteoclastic resorption. Our results demonstrate a view of how myeloma cell growth is halted by P2X7 receptor and the consequences on myeloma–osteoblast and myeloma–osteoclast interaction in vitro. Full article

Programmed Cell Death (PCD) is considered to be a pathological form of cell death when mediated by an intracellular program and it balances cell death with survival of normal cells. Pyroptosis, a type of PCD, is induced by the inflammatory caspase cleavage of gasdermin D (GSDMD) and apoptotic caspase cleavage of gasdermin E (GSDME). This review aims to summarize the latest molecular mechanisms about pyroptosis mediated by pore-forming GSDMD and GSDME proteins that permeabilize plasma and mitochondrial membrane activating pyroptosis and apoptosis. We also discuss the potentiality of pyroptosis as a therapeutic target in human diseases. Blockade of pyroptosis by compounds can treat inflammatory disease and pyroptosis activation contributes to cancer therapy. Full article

Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord K⁺ (TRESK; K_2P18.1) channel is the only member of the two-pore domain K⁺ (K_2P) channel family that is activated by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and linked to migraines. This study was performed to identify the effect of verapamil, which is an L-type Ca²⁺ channel blocker and a prophylaxis for migraines, on the TRESK channel in trigeminal ganglion (TG) neurons, as well as in a heterologous system. Single-channel and whole-cell currents were recorded in TG neurons and HEK-293 cells transfected with mTRESK using patch-clamping techniques. In TG neurons, changes in [Ca²⁺]_i were measured using the fluo-3-AM Ca²⁺ indicator. Verapamil, nifedipine, and NiCl₂ inhibited the whole-cell currents in HEK-293 cells overexpressing mTRESK with IC₅₀ values of 5.2, 54.3, and >100 μM, respectively. The inhibitory effect of verapamil on TRESK channel was also observed in excised patches. In TG neurons, verapamil (10 μM) inhibited TRESK channel activity by approximately 76%. The TRESK channel activity was not dependent on the presence of extracellular Ca²⁺. In addition, the inhibitory effect of verapamil on the TRESK channel remained despite the absence of extracellular Ca²⁺. These findings show that verapamil inhibits the TRESK current independently of the blockade of Ca²⁺ influx in TG neurons. Verapamil will be able to exert its pharmacological effects by modulating TRESK, as well as Ca²⁺ influx, in TG neurons in vitro. We suggest that verapamil could be used as an inhibitor for identifying TRESK channel in TG neurons. Full article

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms. Full article

The Staphylococcus aureus toxin, α-hemolysin, is an important and well-studied virulence factor in staphylococcal infection. It is a soluble monomeric protein that, once secreted by the bacterium, forms a heptameric pore in the membrane of a broad range of host cell types. Hemolysin was recently discovered to bind and activate a disintegrin and metalloprotease 10 (ADAM10). In epithelial and endothelial cells, ADAM10 activation is required for the toxin’s activity against these cells. In host monocytic cells, α-hemolysin activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome leading to production of pro-inflammatory cytokines and cell death. We now show that ADAM10 is critical for α-hemolysin-mediated activation of the NLRP3 inflammasome in human monocytes as siRNA knockdown or chemical blockade of ADAM10-α-hemolysin interaction leads to diminished inflammasome activation and cell death by reducing the available ADAM10 on the cell surface. Unlike epithelial cell and endothelial cell damage, which requires α-hemolysin induced ADAM10 activation, ADAM10 protease activity was not required for NLRP3 inflammasome activation. This work confirms the importance of ADAM10 in immune activation by α-hemolysin, but indicates that host cell signal induction by the toxin is different between host cell types. Full article

Mitochondrial dysfunction occurs early in the progression of Alzheimer’s disease. Amyloid-β peptide has deleterious effects on mitochondrial function and contributes to energy failure, respiratory chain impairment, neuronal apoptosis, and generation of reactive oxygen species in Alzheimer’s disease. The mechanisms underlying amyloid-β induced mitochondrial stress remain unclear. Emerging evidence indicates that mitochondrial permeability transition pore is important for maintenance of mitochondrial and neuronal function in aging and neurodegenerative disease. Cyclophilin D (Cyp D) plays a central role in opening mitochondrial permeability transition pores, ultimately leading to cell death. Interaction of amyloid-β with cyclophilin D triggers or enhances the formation of mitochondrial permeability transition pores, consequently exacerbating mitochondrial and neuronal dysfunction, as shown by decreased mitochondrial membrane potential, impaired mitochondrial respiration function, and increased oxidative stress and cytochrome c release. Blockade of cyclophilin D by genetic abrogation or pharmacologic inhibition protects mitochondria and neurons from amyloid-β induced toxicity, suggesting that cyclophilin D dependent mitochondrial transition pores are a therapeutic target for Alzheimer’s disease. Full article