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17 pages, 3081 KiB

Open AccessArticle

Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells

by Ekaterina N. Lyukmanova, Maxim M. Zaigraev, Dmitrii S. Kulbatskii, Aizek B. Isaev, Ilya D. Kukushkin, Maxim L. Bychkov, Mikhail A. Shulepko, Anton O. Chugunov and Mikhail P. Kirpichnikov

Toxins 2023, 15(10), 612; https://doi.org/10.3390/toxins15100612 - 13 Oct 2023

Viewed by 1302

Abstract

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for [...] Read more.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the ‘head’ loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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24 pages, 8927 KiB

Open AccessFeature PaperArticle

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer

by Ekaterina N. Lyukmanova, Pavel A. Mironov, Dmitrii S. Kulbatskii, Mikhail A. Shulepko, Alexander S. Paramonov, Elizaveta M. Chernaya, Yulia A. Logashina, Yaroslav A. Andreev, Mikhail P. Kirpichnikov and Zakhar O. Shenkarev

Toxins 2023, 15(6), 378; https://doi.org/10.3390/toxins15060378 - 03 Jun 2023

Cited by 3 | Viewed by 1969

Abstract

Phα1β (PnTx3–6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (Ca_V2.2) and TRPA1. In animal models, Phα1β administration reduces both acute and chronic pain. [...] Read more.

Phα1β (PnTx3–6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (Ca_V2.2) and TRPA1. In animal models, Phα1β administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1β and its ¹⁵N-labeled analogue. Spatial structure and dynamics of Phα1β were determined via NMR spectroscopy. The N-terminal domain (Ala1–Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41–Cys52) stapled to ICK by two disulfides exhibits the µs–ms time-scale fluctuations. The Phα1β structure with the disulfide bond patterns Cys1–5, Cys2–7, Cys3–12, Cys4–10, Cys6–11, Cys8–9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1β has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1β significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1β as a gating modifier toxin, probably interacting with S1–S4 gating domains from a membrane-bound state. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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20 pages, 2899 KiB

Open AccessArticle

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica

by Irina N. Gladkikh, Anna A. Klimovich, Rimma S. Kalina, Yulia V. Kozhevnikova, Timur A. Khasanov, Dmitry I. Osmakov, Sergey G. Koshelev, Margarita M. Monastyrnaya, Yaroslav A. Andreev, Elena V. Leychenko and Sergey A. Kozlov

Toxins 2023, 15(5), 341; https://doi.org/10.3390/toxins15050341 - 15 May 2023

Cited by 1 | Viewed by 1573

Abstract

Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone [...] Read more.

Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone toxins, native Hmg 1b-2 and recombinant Hmg 1b-4, both related to APETx-like peptides, inhibited the transient current component of human ASIC3-Δ20 expressed in Xenopus laevis oocytes, but only Hmg 1b-2 inhibited the rat ASIC3 transient current. The Hmg 1b-4 action on rASIC3 as a potentiator was confirmed once again. Both peptides are non-toxic molecules for rodents. In open field and elevated plus maze tests, Hmg 1b-2 had more of an excitatory effect and Hmg 1b-4 had more of an anxiolytic effect on mouse behavior. The analgesic activity of peptides was similar and comparable to diclofenac activity in an acid-induced muscle pain model. In models of acute local inflammation induced by λ-carrageenan or complete Freund’s adjuvant, Hmg 1b-4 had more pronounced and statistically significant anti-inflammatory effects than Hmg 1b-2. It exceeded the effect of diclofenac and, at a dose of 0.1 mg/kg, reduced the volume of the paw almost to the initial volume. Our data highlight the importance of a comprehensive study of novel ASIC-targeting ligands, and in particular, peptide toxins, and present the slightly different biological activity of the two similar toxins. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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18 pages, 3019 KiB

Open AccessArticle

Anti-Inflammatory Activity of 1,4-Naphthoquinones Blocking P2X7 Purinergic Receptors in RAW 264.7 Macrophage Cells

by Sergei A. Kozlovskiy, Evgeny A. Pislyagin, Ekaterina S. Menchinskaya, Ekaterina A. Chingizova, Yuriy E. Sabutski, Sergey G. Polonik, Galina N. Likhatskaya and Dmitry L. Aminin

Toxins 2023, 15(1), 47; https://doi.org/10.3390/toxins15010047 - 05 Jan 2023

Cited by 6 | Viewed by 1556

Abstract

P2X7 receptors are ligand-gated ion channels activated by ATP and play a significant role in cellular immunity. These receptors are considered as a potential therapeutic target for the treatment of multiple inflammatory diseases. In the present work, using spectrofluorimetry, spectrophotometry, Western blotting and [...] Read more.

P2X7 receptors are ligand-gated ion channels activated by ATP and play a significant role in cellular immunity. These receptors are considered as a potential therapeutic target for the treatment of multiple inflammatory diseases. In the present work, using spectrofluorimetry, spectrophotometry, Western blotting and ELISA approaches, the ability of 1,4-naphthoquinone thioglucoside derivatives, compounds U-286 and U-548, to inhibit inflammation induced by ATP/LPS in RAW 264.7 cells via P2X7 receptors was demonstrated. It has been established that the selected compounds were able to inhibit ATP-induced calcium influx and the production of reactive oxygen species, and they also exhibited pronounced antioxidant activity in mouse brain homogenate. In addition, compounds U-286 and U-548 decreased the LPS-induced activity of the COX-2 enzyme, the release of pro-inflammatory cytokines TNF-α and IL-1β in RAW 264.7 cells, and significantly protected macrophage cells against the toxic effects of ATP and LPS. This study highlights the use of 1,4-naphthoquinones as promising purinergic P2X7 receptor antagonists with anti-inflammatory activity. Based on the data obtained, studied synthetic 1,4-NQs can be considered as potential scaffolds for the development of new anti-inflammatory and analgesic drugs. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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18 pages, 3895 KiB

Open AccessFeature PaperArticle

Peptides from the Sea Anemone Metridium senile with Modified Inhibitor Cystine Knot (ICK) Fold Inhibit Nicotinic Acetylcholine Receptors

by Igor E. Kasheverov, Yulia A. Logashina, Fedor D. Kornilov, Vladislav A. Lushpa, Ekaterina E. Maleeva, Yuliya V. Korolkova, Jinpeng Yu, Xiaopeng Zhu, Dongting Zhangsun, Sulan Luo, Klara Stensvåg, Denis S. Kudryavtsev, Konstantin S. Mineev and Yaroslav A. Andreev

Toxins 2023, 15(1), 28; https://doi.org/10.3390/toxins15010028 - 30 Dec 2022

Cited by 3 | Viewed by 2159

Abstract

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological [...] Read more.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1–Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1–Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1–Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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17 pages, 5050 KiB

Open AccessArticle

Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells

by Nikita A. Orlov, Anastasia A. Ignatova, Elena V. Kryukova, Sergey A. Yakimov, Mikhail P. Kirpichnikov, Oksana V. Nekrasova and Alexey V. Feofanov

Toxins 2022, 14(12), 858; https://doi.org/10.3390/toxins14120858 - 05 Dec 2022

Cited by 2 | Viewed by 1572

Abstract

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study [...] Read more.

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels’ structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the “mix and measure”, format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K⁺-conducting pore, and analyzing their affinity. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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15 pages, 2165 KiB

Open AccessArticle

Characterization of a Family of Scorpion Toxins Modulating Ca²⁺-Activated Cl⁻ Current in Vascular Myocytes

by Jean-Luc Morel, Nathalie Mokrzycki, Guy Lippens, Hervé Drobecq, Pierre Sautière and Michel Hugues

Toxins 2022, 14(11), 780; https://doi.org/10.3390/toxins14110780 - 10 Nov 2022

Cited by 2 | Viewed by 1410

Abstract

The pharmacology of calcium-activated chloride current is not well developed. Peptides from scorpion venom present potent pharmacological actions on ionic conductance used to characterize the function of channels but can also be helpful to develop organic pharmacological tools. Using electrophysiological recording coupled with [...] Read more.

The pharmacology of calcium-activated chloride current is not well developed. Peptides from scorpion venom present potent pharmacological actions on ionic conductance used to characterize the function of channels but can also be helpful to develop organic pharmacological tools. Using electrophysiological recording coupled with calcium measurement, we tested the potent effect of peptides extracted from Leuirus quinquestratus quinquestratus venom on the calcium-activated chloride current expressed in smooth muscle cells freshly dissociated from rat portal veins. We identified one peptide which selectively inhibited the chloride conductance without effects on either calcium signaling or calcium and potassium currents expressed in this cell type. The synthetic peptide had the same affinity, but the chemical modification of the amino acid sequence altered the efficiency to inhibit the calcium-activated chloride conductance. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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15 pages, 2237 KiB

Open AccessArticle

The Contribution of Phospholipase A₂ and Metalloproteinases to the Synergistic Action of Viper Venom on the Bioenergetic Profile of Vero Cells

by Naira Ayvazyan, Gevorg Ghukasyan, Lusine Ghulikyan, Gayane Kirakosyan, Gohar Sevoyan, Armen Voskanyan and Zaruhi Karabekyan

Toxins 2022, 14(11), 724; https://doi.org/10.3390/toxins14110724 - 23 Oct 2022

Cited by 3 | Viewed by 1433

Abstract

Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation [...] Read more.

Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation calls for critical review and analysis. The epithelium is a primary target for injected viper venom’s toxic substances, and therefore, is a focus in modern toxinology. We used the Vero epithelial cell line as a model to compare the actions of a crude Macrovipera lebetina obtusa (Levantine viper) venom with the actions of the same venom with two key enzymatic components inhibited (specifically, phospholipase A2 (PLA2) and metalloproteinases) in the bioenergetic cellular response, i.e., oxygen uptake and reactive oxygen species generation. In addition to the rate of free-radical oxidation and lipid peroxidation, we measured real-time mitochondrial respiration (based on the oxygen consumption rate) and glycolysis (based on the extracellular acidification rate) using a Seahorse analyzer. Our data show that viper venom drives an increase in both glycolysis and respiration in Vero cells, while the blockage of PLA2 or/and metalloproteinases affects only the rates of the oxidative phosphorylation. PLA2-blocking in venom also increases cytotoxic activity and the overproduction of reactive oxygen species. These data show that certain components of the venom may have a different effect within the venom cocktail other than the purified enzymes due to the synergy of the venom components. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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20 pages, 4203 KiB

Open AccessArticle

Nicotinic Acetylcholine Receptors Are Novel Targets of APETx-like Toxins from the Sea Anemone Heteractis magnifica

by Rimma S. Kalina, Igor E. Kasheverov, Sergey G. Koshelev, Oksana V. Sintsova, Steve Peigneur, Ernesto Lopes Pinheiro-Junior, Roman S. Popov, Victoria E. Chausova, Margarita M. Monastyrnaya, Pavel S. Dmitrenok, Marina P. Isaeva, Jan Tytgat, Sergey A. Kozlov, Emma P. Kozlovskaya, Elena V. Leychenko and Irina N. Gladkikh

Toxins 2022, 14(10), 697; https://doi.org/10.3390/toxins14100697 - 11 Oct 2022

Cited by 5 | Viewed by 1574

Abstract

The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels, provide cholinergic signaling, and are modulated by various venom toxins and drugs in addition to neurotransmitters. Here, four APETx-like toxins, including two new toxins, named Hmg 1b-2 Met_ox and Hmg 1b-5, were [...] Read more.

The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels, provide cholinergic signaling, and are modulated by various venom toxins and drugs in addition to neurotransmitters. Here, four APETx-like toxins, including two new toxins, named Hmg 1b-2 Met_ox and Hmg 1b-5, were isolated from the sea anemone Heteractis magnifica and characterized as novel nAChR ligands and acid-sensing ion channel (ASIC) modulators. All peptides competed with radiolabeled α-bungarotoxin for binding to Torpedo californica muscle-type and human α7 nAChRs. Hmg 1b-2 potentiated acetylcholine-elicited current in human α7 receptors expressed in Xenopus laevis oocytes. Moreover, the multigene family coding APETx-like peptides library from H. magnifica was described and in silico surface electrostatic potentials of novel peptides were analyzed. To explain the 100% identity of some peptide isoforms between H. magnifica and H. crispa, 18S rRNA, COI, and ITS analysis were performed. It has been shown that the sea anemones previously identified by morphology as H. crispa belong to the species H. magnifica. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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Review

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35 pages, 4230 KiB

Open AccessReview

Snake Venom: A Promising Source of Neurotoxins Targeting Voltage-Gated Potassium Channels

by Altaf K. AlShammari, Tarek Mohamed Abd El-Aziz and Ahmed Al-Sabi

Toxins 2024, 16(1), 12; https://doi.org/10.3390/toxins16010012 - 25 Dec 2023

Viewed by 1781

Abstract

The venom derived from various sources of snakes represents a vast collection of predominantly protein-based toxins that exhibit a wide range of biological actions, including but not limited to inflammation, pain, cytotoxicity, cardiotoxicity, and neurotoxicity. The venom of a particular snake species is [...] Read more.

The venom derived from various sources of snakes represents a vast collection of predominantly protein-based toxins that exhibit a wide range of biological actions, including but not limited to inflammation, pain, cytotoxicity, cardiotoxicity, and neurotoxicity. The venom of a particular snake species is composed of several toxins, while the venoms of around 600 venomous snake species collectively encompass a substantial reservoir of pharmacologically intriguing compounds. Despite extensive research efforts, a significant portion of snake venoms remains uncharacterized. Recent findings have demonstrated the potential application of neurotoxins derived from snake venom in selectively targeting voltage-gated potassium channels (Kv). These neurotoxins include BPTI-Kunitz polypeptides, PLA₂ neurotoxins, CRISPs, SVSPs, and various others. This study provides a comprehensive analysis of the existing literature on the significance of Kv channels in various tissues, highlighting their crucial role as proteins susceptible to modulation by diverse snake venoms. These toxins have demonstrated potential as valuable pharmacological resources and research tools for investigating the structural and functional characteristics of Kv channels. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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21 pages, 1899 KiB

Open AccessReview

Scorpion Peptides and Ion Channels: An Insightful Review of Mechanisms and Drug Development

by Lais Campelo Mendes, Gabriela Magnólia Melo Viana, Ana Leonor Abrahão Nencioni, Daniel Carvalho Pimenta and Emidio Beraldo-Neto

Toxins 2023, 15(4), 238; https://doi.org/10.3390/toxins15040238 - 24 Mar 2023

Cited by 6 | Viewed by 2584

Abstract

The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and [...] Read more.

The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and any disturbance in their activity can result in channelopathies, which can lead to various diseases such as autoimmune, cardiovascular, immunological, neurological, and neoplastic conditions. Given the importance of ion channels, scorpion peptides represent a valuable resource for developing drugs with targeted specificity for these channels. This review provides a comprehensive overview of the structure and classification of ion channels, the action of scorpion toxins on these channels, and potential avenues for future research. Overall, this review highlights the significance of scorpion venom as a promising source for discovering novel drugs with therapeutic potential for treating channelopathies. Full article

(This article belongs to the Special Issue Ion Channels, Venom, and Toxins)

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