The Experts below are selected from a list of 234 Experts worldwide ranked by ideXlab platform
Vincent Seutin - One of the best experts on this subject based on the ideXlab platform.
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the interactions of Apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium activated potassium sk channels
Biochemical Pharmacology, 2013Co-Authors: Sebastien Dilly, Vincent Seutin, Fabian Philippart, Cedric Lamy, Sylvie Poncin, Dirk J Snyders, Jeanfrancois LiegeoisAbstract:Abstract Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker Apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of Apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by Apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by Apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of Apamin with its target.
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crucial role of a shared extracellular loop in Apamin sensitivity and maintenance of pore shape of small conductance calcium activated potassium sk channels
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kate L. Weatherall, Vincent Seutin, Jeanfrancois Liegeois, Neil V. MarrionAbstract:Activation of small-conductance calcium (Ca2+)-dependent potassium (KCa2) channels (herein called “SK”) produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1–3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker Apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest Apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3–S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by Apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by Apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3–S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with Apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.
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allosteric block of kca2 channels by Apamin
Journal of Biological Chemistry, 2010Co-Authors: Cedric Lamy, Vincent Seutin, Kate L. Weatherall, Jeanfrancois Liegeois, Samuel J Goodchild, David E Jane, Neil V. MarrionAbstract:Abstract Activation of small conductance calcium-activated potassium (KCa2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin Apamin, but the mechanism of block is not understood. For example, Apamin binds to both KCa2.2 and KCa2.3 with the same high affinity (KD ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC50 values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that Apamin blocks KCa2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by Apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with Apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that Apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.
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metaplastic effect of Apamin on ltp and paired pulse facilitation
Learning & Memory, 2007Co-Authors: Brigitte Capron, Vincent Seutin, Coralie Sclavons, Jeanfrancois Liegeois, Emile GodauxAbstract:In area CA1 of hippocampal slices, a single 1-sec train of 100-Hz stimulation generally triggers a short-lasting long-term potentiation (S-LTP) of 1-2 h. Here, we found that when such a train was applied 45 min after application of the small conductance Ca2+-activated K+ (SK) channel blocker Apamin, it induced a long-lasting LTP (L-LTP) of several hours, instead of an S-LTP. Apamin-induced SK channel blockage is known to resist washing. Nevertheless, the aforementioned effect is not a mere delayed effect; it is metaplastic. Indeed, when a single train was delivered to the Schaffer's collaterals during Apamin application, it induced an S-LTP, like in the control situation. At the moment of this LTP induction (15th min of Apamin application), the SK channel blockage was nevertheless complete. Indeed, at that time, under the influence of Apamin, the amplitude of the series of field excitatory postsynaptic potentials (fEPSPs) triggered by a stimulation train was increased. We found that the metaplastic effect of Apamin on LTP was crucially dependent on the NO-synthase pathway, whereas the efficacy of the NMDA receptors was not modified at the time of its occurrence. We also found that Apamin produced an increase in paired-pulse facilitation not during, but after, the application of the drug. Finally, we found that the induction of each of these two metaplastic phenomena was mediated by NMDA receptors. A speculative unitary hypothesis to explain these phenomena is proposed.
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bicuculline methiodide potentiates nmda dependent burst firing in rat dopamine neurons by blocking Apamin sensitive ca2 activated k currents
Neuroscience Letters, 1997Co-Authors: Steven W Johnson, Vincent SeutinAbstract:Abstract Apamin, a bee venom toxin which blocks a Ca 2+ -dependent K + current, potentiates N -methyl- d -aspartate (NMDA)-induced burst firing in dopamine neurons. We now report that burst firing is also potentiated by an Apamin-like effect of bicuculline methiodide (BMI) at the same concentration (30 μ M) which blocks GABA A receptors in vitro. Using microelectrodes to record intracellularly from rat dopamine neurons in the midbrain slice, BMI reduced the Apamin-sensitive afterhyperpolarization in all cells tested. BMI also mimicked Apamin (100 nM) by potentiating burst firing produced by a concentration of NMDA (10 μ M) which is too low to evoke burst firing when perfused alone. When recording under voltage-clamp, both BMI and Apamin reduced a depolarization-activated outward current which was also sensitive to perfusate containing no-added Ca 2+ . Although picrotoxin (100 μ M) and bicuculline free base (30 μ M) blocked the inhibition of firing produced by the GABA A agonist isoguvacine (100 μ M), neither had Apamin-like effects. We conclude that BMI potentiates burst firing by blocking an Apamin-sensitive Ca 2+ -activated K + current.
Kwankyu Park - One of the best experts on this subject based on the ideXlab platform.
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Therapeutic Effects of Apamin as a Bee Venom Component for Non-Neoplastic Disease.
Toxins, 2020Co-Authors: Hyemin Gu, Kwankyu ParkAbstract:Bee venom is a natural toxin produced by honeybees and plays an important role in defending bee colonies. Bee venom has several kinds of peptides, including melittin, Apamin, adolApamine, and mast cell degranulation peptides. Apamin accounts for about 2%-3% dry weight of bee venom and is a peptide neurotoxin that contains 18 amino acid residues that are tightly crosslinked by two disulfide bonds. It is well known for its pharmacological functions, which irreversibly block Ca2+-activated K+ (SK) channels. Apamin regulates gene expression in various signal transduction pathways involved in cell development. The aim of this study was to review the current understanding of Apamin in the treatment of apoptosis, fibrosis, and central nervous system diseases, which are the pathological processes of various diseases. Apamin's potential therapeutic and pharmacological applications are also discussed.
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Apamin suppresses biliary fibrosis and activation of hepatic stellate cells
International Journal of Molecular Medicine, 2017Co-Authors: Hyunjin An, Yoonyub Park, Kyung Duck Park, Kwankyu ParkAbstract:: Cholestatic liver disease is characterized by the progressive destruction of biliary epithelial cells (BECs) followed by fibrosis, cirrhosis and liver failure. Activated hepatic stellate cells (HSCs) and portal fibroblasts are the major cellular effectors of enhanced collagen deposition in biliary fibrosis. Apamin, an 18 amino acid peptide neurotoxin found in apitoxin (bee venom), is known to block Ca2+-activated K+ channels and prevent carbon tetrachloride-induced liver fibrosis. In the present study, we aimed to ascertain whether Apamin inhibits biliary fibrosis and the proliferation of HSCs. Cholestatic liver fibrosis was established in mouse models with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Cellular assays were performed on HSC-T6 cells (rat immortalized HSCs). DDC feeding led to increased hepatic damage and proinflammtory cytokine levels. Notably, Apamin treatment resulted in decreased liver injury and proinflammatory cytokine levels. Moreover, Apamin suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice. In HSCs, Apamin suppressed activation of HSCs by inhibiting the Smad signaling pathway. These data suggest that Apamin may be a potential therapeutic target in cholestatic liver disease.
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Apamin inhibits tnf α and ifn γ induced inflammatory cytokines and chemokines via suppressions of nf κb signaling pathway and stat in human keratinocytes
Pharmacological Reports, 2017Co-Authors: Hyunjin An, Kyung Duck Park, Migyeong Gwon, Hyemin Gu, Ji Y Park, Kwankyu ParkAbstract:Abstract Background Atopic dermatitis (AD) is identified by an increase in infiltrations of several inflammatory cells including type 2 helper (Th2) lymphocytes. Th2-related chemokines such as thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), and pro-inflammatory cytokines including interleukin (IL)-1β and IL-6 are considered to play a crucial role in AD. Tumor necrosis factor (TNF)-α- and interferon (IFN)-γ induce the inflammatory condition through production of TARC, MDC, IL-1β and IL-6, and activations of related transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT) in keratinocytes. Apamin, a peptide component of bee venom, has been reported its beneficial activities in various diseases. However, anti-inflammatory effects of Apamin on inflammatory condition in keratinocytes have not been explored. Therefore, the present study aimed to demonstrate the anti-inflammatory effect of Apamin on TNF-α- and IFN-γ-induced inflammatory condition in keratinocytes. Methods HaCaT was used as human keratinocytes cell line. Cell Counting Kit-8 was performed to measure a cytotoxicity of Apamin. The effects of Apamin on TNF-α-/IFN-γ-induced inflammatory condition were determined by real-time PCR and Western blot analysis. Further, NF-κB signaling pathways, STAT1, and STAT3 were analyzed by Western blot and immunofluorescence. Results Apamin ameliorated the inflammatory condition through suppression of Th2-related chemokines and pro-inflammatory cytokines. Further, Apamin down-regulated the activations of NF-κB signaling pathways and STATs in HaCaT cells. Conclusions These results suggest that Apamin has therapeutic effect on AD through improvement of inflammatory condition.
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Apamin inhibits pdgf bb induced vascular smooth muscle cell proliferation and migration through suppressions of activated akt and erk signaling pathway
Vascular Pharmacology, 2015Co-Authors: Hyunjin An, Yoonyub Park, Kwankyu ParkAbstract:Abstract The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca 2 + -activated K + channels. However, the effects of Apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of Apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, Apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21 Cip1 in PDGF-BB-induced VSMC. Moreover, Apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that Apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, Apamin may be a promising candidate for the therapy of atherosclerosis.
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Apamin inhibits hepatic fibrosis through suppression of transforming growth factor β1 induced hepatocyte epithelial mesenchymal transition
Biochemical and Biophysical Research Communications, 2014Co-Authors: Hyunjin An, Kwankyu ParkAbstract:Abstract Apamin is an integral part of bee venom, as a peptide component. It has long been known as a highly selective block Ca2+-activated K+ (SK) channels. However, the cellular mechanism and anti-fibrotic effect of Apamin in TGF-β1-induced hepatocytes have not been explored. In the present study, we investigated the anti-fibrosis or anti-EMT mechanism by examining the effect of Apamin on TGF-β1-induced hepatocytes. AML12 cells were seeded at ∼60% confluence in complete growth medium. Twenty-four hours later, the cells were changed to serum free medium containing the indicated concentrations of Apamin. After 30 min, the cells were treated with 2 ng/ml of TGF-β1 and co-cultured for 48 h. Also, we investigated the effects of Apamin on the CCl4-induced liver fibrosis animal model. Treatment of AML12 cells with 2 ng/ml of TGF-β1 resulted in loss of E-cadherin protein at the cell–cell junctions and concomitant increased expression of vimentin. In addition, phosphorylation levels of ERK1/2, Akt, Smad2/3 and Smad4 were increased by TGF-β1 stimulation. However, cells treated concurrently with TGF-β1 and Apamin retained high levels of localized expression of E-cadherin and showed no increase in vimentin. Specifically, treatment with 2 μg/ml of Apamin almost completely blocked the phosphorylation of ERK1/2, Akt, Smad2/3 and Smad4 in AML12 cells. In addition, Apamin exhibited prevention of pathological changes in the CCl4-injected animal models. These results demonstrate the potential of Apamin for the prevention of EMT progression induced by TGF-β1 in vitro and CCl4-injected in vivo.
Jeanfrancois Liegeois - One of the best experts on this subject based on the ideXlab platform.
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the interactions of Apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium activated potassium sk channels
Biochemical Pharmacology, 2013Co-Authors: Sebastien Dilly, Vincent Seutin, Fabian Philippart, Cedric Lamy, Sylvie Poncin, Dirk J Snyders, Jeanfrancois LiegeoisAbstract:Abstract Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker Apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of Apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by Apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by Apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of Apamin with its target.
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crucial role of a shared extracellular loop in Apamin sensitivity and maintenance of pore shape of small conductance calcium activated potassium sk channels
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kate L. Weatherall, Vincent Seutin, Jeanfrancois Liegeois, Neil V. MarrionAbstract:Activation of small-conductance calcium (Ca2+)-dependent potassium (KCa2) channels (herein called “SK”) produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1–3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker Apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest Apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3–S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by Apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by Apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3–S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with Apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.
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allosteric block of kca2 channels by Apamin
Journal of Biological Chemistry, 2010Co-Authors: Cedric Lamy, Vincent Seutin, Kate L. Weatherall, Jeanfrancois Liegeois, Samuel J Goodchild, David E Jane, Neil V. MarrionAbstract:Abstract Activation of small conductance calcium-activated potassium (KCa2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin Apamin, but the mechanism of block is not understood. For example, Apamin binds to both KCa2.2 and KCa2.3 with the same high affinity (KD ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC50 values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that Apamin blocks KCa2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by Apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with Apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that Apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.
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metaplastic effect of Apamin on ltp and paired pulse facilitation
Learning & Memory, 2007Co-Authors: Brigitte Capron, Vincent Seutin, Coralie Sclavons, Jeanfrancois Liegeois, Emile GodauxAbstract:In area CA1 of hippocampal slices, a single 1-sec train of 100-Hz stimulation generally triggers a short-lasting long-term potentiation (S-LTP) of 1-2 h. Here, we found that when such a train was applied 45 min after application of the small conductance Ca2+-activated K+ (SK) channel blocker Apamin, it induced a long-lasting LTP (L-LTP) of several hours, instead of an S-LTP. Apamin-induced SK channel blockage is known to resist washing. Nevertheless, the aforementioned effect is not a mere delayed effect; it is metaplastic. Indeed, when a single train was delivered to the Schaffer's collaterals during Apamin application, it induced an S-LTP, like in the control situation. At the moment of this LTP induction (15th min of Apamin application), the SK channel blockage was nevertheless complete. Indeed, at that time, under the influence of Apamin, the amplitude of the series of field excitatory postsynaptic potentials (fEPSPs) triggered by a stimulation train was increased. We found that the metaplastic effect of Apamin on LTP was crucially dependent on the NO-synthase pathway, whereas the efficacy of the NMDA receptors was not modified at the time of its occurrence. We also found that Apamin produced an increase in paired-pulse facilitation not during, but after, the application of the drug. Finally, we found that the induction of each of these two metaplastic phenomena was mediated by NMDA receptors. A speculative unitary hypothesis to explain these phenomena is proposed.
Neil V. Marrion - One of the best experts on this subject based on the ideXlab platform.
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Contribution of S3-S4 Extracellular Loop Residues to Block of Kca2 Channels by Apamin
Biophysical Journal, 2020Co-Authors: Kate L. Weatherall, Neil V. MarrionAbstract:Small conductance Ca2+-activated K+ (KCa2) channels are widely distributed within the CNS and peripheral tissues. The cloning of these channels revealed three subtypes, each displaying a different sensitivity to block by the KCa2 selective toxin Apamin. Mutation studies of hKCa2.1 and rKCa2.2 have indicated the importance of particular residues in both the channel pore and S3-S4 extracellular loop for block by Apamin.It has been reported that mutation of threonine (T)216 within the S3-S4 loop of hKCa2.1 to the corresponding serine (S) in rKCa2.2 resulted in a current that was more sensitive to block by Apamin (Nolting et al, 2007; JBC 282, 3478). We have further investigated the residues in this extracellular loop region that contribute to block by Apamin, d-tubocurarine (dTC) and tetraethylammonium (TEA). Block of expressed KCa2 channel current was assessed using outside-out macropatches, with current activated by 1 μM intracellular Ca2+. Mutation S245 to T, to mirror the previously reported mutation of hKCa2.1(T216S), resulted in a reduction in the sensitivity to Apamin and no change in the sensitivity to TEA and dTC. Double point mutation of the loop YA246/7 of rKCa2.2 to the corresponding LV of the Apamin-insensitive rKCa2.1, abolished block by Apamin, reduced sensitivity to dTC sensitivity, but did not affect sensitivity to TEA. In contrast, generation of KCa2.2(Y246L) reduced sensitivity to block by Apamin, but did not change sensitivity to dTC. These data suggest that additional residues within the S3-S4 extracellular loop contribute to the high sensitivity to block by Apamin exhibited by KCa2.2.
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crucial role of a shared extracellular loop in Apamin sensitivity and maintenance of pore shape of small conductance calcium activated potassium sk channels
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kate L. Weatherall, Vincent Seutin, Jeanfrancois Liegeois, Neil V. MarrionAbstract:Activation of small-conductance calcium (Ca2+)-dependent potassium (KCa2) channels (herein called “SK”) produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1–3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker Apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest Apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3–S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by Apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by Apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3–S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with Apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.
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allosteric block of kca2 channels by Apamin
Journal of Biological Chemistry, 2010Co-Authors: Cedric Lamy, Vincent Seutin, Kate L. Weatherall, Jeanfrancois Liegeois, Samuel J Goodchild, David E Jane, Neil V. MarrionAbstract:Abstract Activation of small conductance calcium-activated potassium (KCa2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin Apamin, but the mechanism of block is not understood. For example, Apamin binds to both KCa2.2 and KCa2.3 with the same high affinity (KD ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC50 values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that Apamin blocks KCa2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by Apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with Apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that Apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.
Hyunjin An - One of the best experts on this subject based on the ideXlab platform.
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Apamin suppresses biliary fibrosis and activation of hepatic stellate cells
International Journal of Molecular Medicine, 2017Co-Authors: Hyunjin An, Yoonyub Park, Kyung Duck Park, Kwankyu ParkAbstract:: Cholestatic liver disease is characterized by the progressive destruction of biliary epithelial cells (BECs) followed by fibrosis, cirrhosis and liver failure. Activated hepatic stellate cells (HSCs) and portal fibroblasts are the major cellular effectors of enhanced collagen deposition in biliary fibrosis. Apamin, an 18 amino acid peptide neurotoxin found in apitoxin (bee venom), is known to block Ca2+-activated K+ channels and prevent carbon tetrachloride-induced liver fibrosis. In the present study, we aimed to ascertain whether Apamin inhibits biliary fibrosis and the proliferation of HSCs. Cholestatic liver fibrosis was established in mouse models with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Cellular assays were performed on HSC-T6 cells (rat immortalized HSCs). DDC feeding led to increased hepatic damage and proinflammtory cytokine levels. Notably, Apamin treatment resulted in decreased liver injury and proinflammatory cytokine levels. Moreover, Apamin suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice. In HSCs, Apamin suppressed activation of HSCs by inhibiting the Smad signaling pathway. These data suggest that Apamin may be a potential therapeutic target in cholestatic liver disease.
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Apamin inhibits tnf α and ifn γ induced inflammatory cytokines and chemokines via suppressions of nf κb signaling pathway and stat in human keratinocytes
Pharmacological Reports, 2017Co-Authors: Hyunjin An, Kyung Duck Park, Migyeong Gwon, Hyemin Gu, Ji Y Park, Kwankyu ParkAbstract:Abstract Background Atopic dermatitis (AD) is identified by an increase in infiltrations of several inflammatory cells including type 2 helper (Th2) lymphocytes. Th2-related chemokines such as thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), and pro-inflammatory cytokines including interleukin (IL)-1β and IL-6 are considered to play a crucial role in AD. Tumor necrosis factor (TNF)-α- and interferon (IFN)-γ induce the inflammatory condition through production of TARC, MDC, IL-1β and IL-6, and activations of related transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT) in keratinocytes. Apamin, a peptide component of bee venom, has been reported its beneficial activities in various diseases. However, anti-inflammatory effects of Apamin on inflammatory condition in keratinocytes have not been explored. Therefore, the present study aimed to demonstrate the anti-inflammatory effect of Apamin on TNF-α- and IFN-γ-induced inflammatory condition in keratinocytes. Methods HaCaT was used as human keratinocytes cell line. Cell Counting Kit-8 was performed to measure a cytotoxicity of Apamin. The effects of Apamin on TNF-α-/IFN-γ-induced inflammatory condition were determined by real-time PCR and Western blot analysis. Further, NF-κB signaling pathways, STAT1, and STAT3 were analyzed by Western blot and immunofluorescence. Results Apamin ameliorated the inflammatory condition through suppression of Th2-related chemokines and pro-inflammatory cytokines. Further, Apamin down-regulated the activations of NF-κB signaling pathways and STATs in HaCaT cells. Conclusions These results suggest that Apamin has therapeutic effect on AD through improvement of inflammatory condition.
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Apamin inhibits pdgf bb induced vascular smooth muscle cell proliferation and migration through suppressions of activated akt and erk signaling pathway
Vascular Pharmacology, 2015Co-Authors: Hyunjin An, Yoonyub Park, Kwankyu ParkAbstract:Abstract The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca 2 + -activated K + channels. However, the effects of Apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of Apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, Apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21 Cip1 in PDGF-BB-induced VSMC. Moreover, Apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that Apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, Apamin may be a promising candidate for the therapy of atherosclerosis.
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Apamin inhibits hepatic fibrosis through suppression of transforming growth factor β1 induced hepatocyte epithelial mesenchymal transition
Biochemical and Biophysical Research Communications, 2014Co-Authors: Hyunjin An, Kwankyu ParkAbstract:Abstract Apamin is an integral part of bee venom, as a peptide component. It has long been known as a highly selective block Ca2+-activated K+ (SK) channels. However, the cellular mechanism and anti-fibrotic effect of Apamin in TGF-β1-induced hepatocytes have not been explored. In the present study, we investigated the anti-fibrosis or anti-EMT mechanism by examining the effect of Apamin on TGF-β1-induced hepatocytes. AML12 cells were seeded at ∼60% confluence in complete growth medium. Twenty-four hours later, the cells were changed to serum free medium containing the indicated concentrations of Apamin. After 30 min, the cells were treated with 2 ng/ml of TGF-β1 and co-cultured for 48 h. Also, we investigated the effects of Apamin on the CCl4-induced liver fibrosis animal model. Treatment of AML12 cells with 2 ng/ml of TGF-β1 resulted in loss of E-cadherin protein at the cell–cell junctions and concomitant increased expression of vimentin. In addition, phosphorylation levels of ERK1/2, Akt, Smad2/3 and Smad4 were increased by TGF-β1 stimulation. However, cells treated concurrently with TGF-β1 and Apamin retained high levels of localized expression of E-cadherin and showed no increase in vimentin. Specifically, treatment with 2 μg/ml of Apamin almost completely blocked the phosphorylation of ERK1/2, Akt, Smad2/3 and Smad4 in AML12 cells. In addition, Apamin exhibited prevention of pathological changes in the CCl4-injected animal models. These results demonstrate the potential of Apamin for the prevention of EMT progression induced by TGF-β1 in vitro and CCl4-injected in vivo.
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Apamin inhibits thp 1 derived macrophage apoptosis via mitochondria related apoptotic pathway
Experimental and Molecular Pathology, 2012Co-Authors: Jihyun Park, Hyunjin An, Kwankyu ParkAbstract:Abstract The development of atherosclerotic lesions is mainly due to macrophage death. The oxidative stresses of monocytes/macrophages play a vital role in the initiation and amplification of atherosclerosis. Apamin, a component of bee venom, exerts an anti-inflammatory effect, and selectively inhibits the Ca 2 + -activated K + channels. The mechanisms involved in the inhibition of macrophage apoptosis have been fully elucidated. We induced oxidized low-density lipoprotein (oxLDL) in THP-1-derived macrophage and studied the effect of Apamin on intercellular lipid levels, mitochondria-related apoptotic pathway and numbers of apoptotic cells. Oil-red O staining indicates that the inhibition of Apamin in the condition significantly prevents intracellular lipid deposition. Treatment with Apamin significantly decreased the apoptotic macrophages by decreasing the expression of pro-apoptotic genes Bax, caspase-3 and PARP protein levels, as well as through increasing expression of anti-apoptotic genes Bcl-2 and Bcl-xL protein levels in the absence and presence of oxLDL. In vivo, with Apamin treatment reduced apoptotic cells death by TUNEL staining. These results indicate that Apamin plays an important role in monocyte/macrophage apoptotic processing, which may provide a potential drug for preventing atherosclerosis.
