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Dalia Gordon - One of the best experts on this subject based on the ideXlab platform.
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mapping the Receptor Site for α scorpion toxins on a na channel voltage sensor
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Jinti Wang, Michel Gurevitz, Roy Kahn, Dalia Gordon, Todd Scheuer, Vladimir Yarovyarovoy, William A CatterallAbstract:The α-scorpions toxins bind to the resting state of Na+ channels and inhibit fast inactivation by interaction with a Receptor Site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ∼73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the Receptor Site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na+ channels and reveals its mode of interaction with a gating modifier toxin.
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Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Biophysical Journal, 2010Co-Authors: Zhongli Zhang, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, Michel GurevitzAbstract:Voltage-gated sodium channels are molecular targets of β-scorpion toxins, which enhance excitability by shifting the voltage dependence of activation to more negative potentials. These effects result from a voltage sensor trapping mechanism, in which toxins trap the voltage sensor in its activated conformation. Determinants of β-scorpion toxin (CssIV) binding and action on sodium channel (Nav1.2) are located in the S1-S2 and S3-S4 extracellular linkers in the voltage-sensing module in domain II. To completely map these regions, we made substitutions for previously unstudied amino acid residues and examined modulation by CssIVE15A, a highly active toxin derivative. Of 11 positions studied in IIS1-S2, only one significantly altered the toxin effect from wild-type by reducing binding to the resting state and almost abolishing trapping activity. In IIS3-S4, five positions surrounding a previously identified key binding determinant, G845, define a hotspot of high impact residues. Three of these substitutions reduced toxin binding and voltage-sensor trapping. The other two, V843A and E844N, increased voltage-sensor trapping approximately 4-fold and decreased apparent EC50. The rate of voltage sensor trapping upon depolarization was unchanged for V843A and increased approximately 2.5-fold for E844N. The rate at which the toxin releases the voltage sensor upon repolarization was increased 2.2-fold for the V843A but was unchanged for E844N. Thus CssIVE15A interacts with a short segment of IIS1-S2 and a broader region of DIIS3-S4. The bidirectional effects of mutations on toxin efficacy suggest that native residues make both positive and negative interactions with the toxin. Substitutions that increase toxin effects do so by increasing affinity of resting channels for the toxin and further increasing the relative affinity of the activated voltage-sensor for the toxin. These results provide further support for the voltage sensor-trapping model.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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the differential preference of scorpion α toxins for insect or mammalian sodium channels implications for improved insect control
Toxicon, 2007Co-Authors: Dalia Gordon, Lior Cohen, Roy Kahn, Izhar Karbat, Ke Dong, Nicolas Gilles, Nitza Ilan, Walter Stuhmer, Jan Tytgat, Michel GurevitzAbstract:Receptor Site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This Site interacts allosterically with other topologically distinct Receptors that bind alkaloids, lypophilic polyether toxins, pyrethroids, and Site-4 scorpion toxins. These features suggest that design of insecticides with specificity for Site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion α-toxins in envenomation and their vast use in the study of channel functions, molecular details on Site-3 are scarce. Scorpion α-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, Receptor Site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion α-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting Receptor Site-3. These details, combined with the variations in allosteric interactions between Site-3 and the other Receptor Sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.
Michel Gurevitz - One of the best experts on this subject based on the ideXlab platform.
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mapping the Receptor Site for α scorpion toxins on a na channel voltage sensor
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Jinti Wang, Michel Gurevitz, Roy Kahn, Dalia Gordon, Todd Scheuer, Vladimir Yarovyarovoy, William A CatterallAbstract:The α-scorpions toxins bind to the resting state of Na+ channels and inhibit fast inactivation by interaction with a Receptor Site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ∼73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the Receptor Site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na+ channels and reveals its mode of interaction with a gating modifier toxin.
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Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Biophysical Journal, 2010Co-Authors: Zhongli Zhang, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, Michel GurevitzAbstract:Voltage-gated sodium channels are molecular targets of β-scorpion toxins, which enhance excitability by shifting the voltage dependence of activation to more negative potentials. These effects result from a voltage sensor trapping mechanism, in which toxins trap the voltage sensor in its activated conformation. Determinants of β-scorpion toxin (CssIV) binding and action on sodium channel (Nav1.2) are located in the S1-S2 and S3-S4 extracellular linkers in the voltage-sensing module in domain II. To completely map these regions, we made substitutions for previously unstudied amino acid residues and examined modulation by CssIVE15A, a highly active toxin derivative. Of 11 positions studied in IIS1-S2, only one significantly altered the toxin effect from wild-type by reducing binding to the resting state and almost abolishing trapping activity. In IIS3-S4, five positions surrounding a previously identified key binding determinant, G845, define a hotspot of high impact residues. Three of these substitutions reduced toxin binding and voltage-sensor trapping. The other two, V843A and E844N, increased voltage-sensor trapping approximately 4-fold and decreased apparent EC50. The rate of voltage sensor trapping upon depolarization was unchanged for V843A and increased approximately 2.5-fold for E844N. The rate at which the toxin releases the voltage sensor upon repolarization was increased 2.2-fold for the V843A but was unchanged for E844N. Thus CssIVE15A interacts with a short segment of IIS1-S2 and a broader region of DIIS3-S4. The bidirectional effects of mutations on toxin efficacy suggest that native residues make both positive and negative interactions with the toxin. Substitutions that increase toxin effects do so by increasing affinity of resting channels for the toxin and further increasing the relative affinity of the activated voltage-sensor for the toxin. These results provide further support for the voltage sensor-trapping model.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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the differential preference of scorpion α toxins for insect or mammalian sodium channels implications for improved insect control
Toxicon, 2007Co-Authors: Dalia Gordon, Lior Cohen, Roy Kahn, Izhar Karbat, Ke Dong, Nicolas Gilles, Nitza Ilan, Walter Stuhmer, Jan Tytgat, Michel GurevitzAbstract:Receptor Site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This Site interacts allosterically with other topologically distinct Receptors that bind alkaloids, lypophilic polyether toxins, pyrethroids, and Site-4 scorpion toxins. These features suggest that design of insecticides with specificity for Site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion α-toxins in envenomation and their vast use in the study of channel functions, molecular details on Site-3 are scarce. Scorpion α-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, Receptor Site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion α-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting Receptor Site-3. These details, combined with the variations in allosteric interactions between Site-3 and the other Receptor Sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.
Izhar Karbat - One of the best experts on this subject based on the ideXlab platform.
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Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Biophysical Journal, 2010Co-Authors: Zhongli Zhang, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, Michel GurevitzAbstract:Voltage-gated sodium channels are molecular targets of β-scorpion toxins, which enhance excitability by shifting the voltage dependence of activation to more negative potentials. These effects result from a voltage sensor trapping mechanism, in which toxins trap the voltage sensor in its activated conformation. Determinants of β-scorpion toxin (CssIV) binding and action on sodium channel (Nav1.2) are located in the S1-S2 and S3-S4 extracellular linkers in the voltage-sensing module in domain II. To completely map these regions, we made substitutions for previously unstudied amino acid residues and examined modulation by CssIVE15A, a highly active toxin derivative. Of 11 positions studied in IIS1-S2, only one significantly altered the toxin effect from wild-type by reducing binding to the resting state and almost abolishing trapping activity. In IIS3-S4, five positions surrounding a previously identified key binding determinant, G845, define a hotspot of high impact residues. Three of these substitutions reduced toxin binding and voltage-sensor trapping. The other two, V843A and E844N, increased voltage-sensor trapping approximately 4-fold and decreased apparent EC50. The rate of voltage sensor trapping upon depolarization was unchanged for V843A and increased approximately 2.5-fold for E844N. The rate at which the toxin releases the voltage sensor upon repolarization was increased 2.2-fold for the V843A but was unchanged for E844N. Thus CssIVE15A interacts with a short segment of IIS1-S2 and a broader region of DIIS3-S4. The bidirectional effects of mutations on toxin efficacy suggest that native residues make both positive and negative interactions with the toxin. Substitutions that increase toxin effects do so by increasing affinity of resting channels for the toxin and further increasing the relative affinity of the activated voltage-sensor for the toxin. These results provide further support for the voltage sensor-trapping model.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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the differential preference of scorpion α toxins for insect or mammalian sodium channels implications for improved insect control
Toxicon, 2007Co-Authors: Dalia Gordon, Lior Cohen, Roy Kahn, Izhar Karbat, Ke Dong, Nicolas Gilles, Nitza Ilan, Walter Stuhmer, Jan Tytgat, Michel GurevitzAbstract:Receptor Site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This Site interacts allosterically with other topologically distinct Receptors that bind alkaloids, lypophilic polyether toxins, pyrethroids, and Site-4 scorpion toxins. These features suggest that design of insecticides with specificity for Site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion α-toxins in envenomation and their vast use in the study of channel functions, molecular details on Site-3 are scarce. Scorpion α-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, Receptor Site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion α-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting Receptor Site-3. These details, combined with the variations in allosteric interactions between Site-3 and the other Receptor Sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.
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x ray structure and mutagenesis of the scorpion depressant toxin lqhit2 reveals key determinants crucial for activity and anti insect selectivity
Journal of Molecular Biology, 2007Co-Authors: Izhar Karbat, Michel Gurevitz, Lior Cohen, Roy Kahn, Dalia Gordon, Michael Turkov, Felix FrolowAbstract:Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for Receptor Site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the Receptor Site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the Receptor Site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.
Lior Cohen - One of the best experts on this subject based on the ideXlab platform.
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Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Biophysical Journal, 2010Co-Authors: Zhongli Zhang, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, Michel GurevitzAbstract:Voltage-gated sodium channels are molecular targets of β-scorpion toxins, which enhance excitability by shifting the voltage dependence of activation to more negative potentials. These effects result from a voltage sensor trapping mechanism, in which toxins trap the voltage sensor in its activated conformation. Determinants of β-scorpion toxin (CssIV) binding and action on sodium channel (Nav1.2) are located in the S1-S2 and S3-S4 extracellular linkers in the voltage-sensing module in domain II. To completely map these regions, we made substitutions for previously unstudied amino acid residues and examined modulation by CssIVE15A, a highly active toxin derivative. Of 11 positions studied in IIS1-S2, only one significantly altered the toxin effect from wild-type by reducing binding to the resting state and almost abolishing trapping activity. In IIS3-S4, five positions surrounding a previously identified key binding determinant, G845, define a hotspot of high impact residues. Three of these substitutions reduced toxin binding and voltage-sensor trapping. The other two, V843A and E844N, increased voltage-sensor trapping approximately 4-fold and decreased apparent EC50. The rate of voltage sensor trapping upon depolarization was unchanged for V843A and increased approximately 2.5-fold for E844N. The rate at which the toxin releases the voltage sensor upon repolarization was increased 2.2-fold for the V843A but was unchanged for E844N. Thus CssIVE15A interacts with a short segment of IIS1-S2 and a broader region of DIIS3-S4. The bidirectional effects of mutations on toxin efficacy suggest that native residues make both positive and negative interactions with the toxin. Substitutions that increase toxin effects do so by increasing affinity of resting channels for the toxin and further increasing the relative affinity of the activated voltage-sensor for the toxin. These results provide further support for the voltage sensor-trapping model.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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the differential preference of scorpion α toxins for insect or mammalian sodium channels implications for improved insect control
Toxicon, 2007Co-Authors: Dalia Gordon, Lior Cohen, Roy Kahn, Izhar Karbat, Ke Dong, Nicolas Gilles, Nitza Ilan, Walter Stuhmer, Jan Tytgat, Michel GurevitzAbstract:Receptor Site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This Site interacts allosterically with other topologically distinct Receptors that bind alkaloids, lypophilic polyether toxins, pyrethroids, and Site-4 scorpion toxins. These features suggest that design of insecticides with specificity for Site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion α-toxins in envenomation and their vast use in the study of channel functions, molecular details on Site-3 are scarce. Scorpion α-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, Receptor Site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion α-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting Receptor Site-3. These details, combined with the variations in allosteric interactions between Site-3 and the other Receptor Sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.
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x ray structure and mutagenesis of the scorpion depressant toxin lqhit2 reveals key determinants crucial for activity and anti insect selectivity
Journal of Molecular Biology, 2007Co-Authors: Izhar Karbat, Michel Gurevitz, Lior Cohen, Roy Kahn, Dalia Gordon, Michael Turkov, Felix FrolowAbstract:Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for Receptor Site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the Receptor Site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the Receptor Site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.
Roy Kahn - One of the best experts on this subject based on the ideXlab platform.
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mapping the Receptor Site for α scorpion toxins on a na channel voltage sensor
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Jinti Wang, Michel Gurevitz, Roy Kahn, Dalia Gordon, Todd Scheuer, Vladimir Yarovyarovoy, William A CatterallAbstract:The α-scorpions toxins bind to the resting state of Na+ channels and inhibit fast inactivation by interaction with a Receptor Site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ∼73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the Receptor Site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na+ channels and reveals its mode of interaction with a gating modifier toxin.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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molecular analysis of the sea anemone toxin av3 reveals selectivity to insects and demonstrates the heterogeneity of Receptor Site 3 on voltage gated na channels
Biochemical Journal, 2007Co-Authors: Yehu Moran, Lior Cohen, Roy Kahn, Izhar Karbat, Dalia Gordon, Michel GurevitzAbstract:Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Navs (voltage-gated Na+ channels) like the structurally dissimilar scorpion α-toxins and type I sea anemone toxins that bind to Receptor Site-3. To examine the potency and mode of interaction of Av3 with insect Navs, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65±0.46 pmol/100 mg), to compete well with the Site-3 toxin LqhαIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (Ki=21.4±7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNav1, but not that of mammalian Navs expressed in Xenopus oocytes. Moreover, like other Site-3 toxins, the activity of Av3 was synergically enhanced by ligands of Receptor Site-4 (e.g. scorpion β-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other Site-3 toxins. These analyses have portrayed a toxin that might interact with Receptor Site-3 in a different fashion compared with other ligands of this Site. This assumption was corroborated by a D1701R mutation in DmNav1, which has been shown to abolish the activity of all other Site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of Receptor Site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
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the differential preference of scorpion α toxins for insect or mammalian sodium channels implications for improved insect control
Toxicon, 2007Co-Authors: Dalia Gordon, Lior Cohen, Roy Kahn, Izhar Karbat, Ke Dong, Nicolas Gilles, Nitza Ilan, Walter Stuhmer, Jan Tytgat, Michel GurevitzAbstract:Receptor Site-3 on voltage-gated sodium channels is targeted by a variety of structurally distinct toxins from scorpions, sea anemones, and spiders whose typical action is the inhibition of sodium current inactivation. This Site interacts allosterically with other topologically distinct Receptors that bind alkaloids, lypophilic polyether toxins, pyrethroids, and Site-4 scorpion toxins. These features suggest that design of insecticides with specificity for Site-3 might be rewarding due to the positive cooperativity with other toxins or insecticidal agents. Yet, despite the central role of scorpion α-toxins in envenomation and their vast use in the study of channel functions, molecular details on Site-3 are scarce. Scorpion α-toxins vary greatly in preference for sodium channels of insects and mammals, and some of them are highly active on insects. This implies that despite its commonality, Receptor Site-3 varies on insect vs. mammalian channels, and that elucidation of these differences could potentially be exploited for manipulation of toxin preference. This review provides current perspectives on (i) the classification of scorpion α-toxins, (ii) their mode of interaction with sodium channels and pharmacological divergence, (iii) molecular details on their bioactive surfaces and differences associated with preference for channel subtypes, as well as (iv) a summary of the present knowledge about elements involved in constituting Receptor Site-3. These details, combined with the variations in allosteric interactions between Site-3 and the other Receptor Sites on insect and mammalian sodium channels, may be useful in new strategies of insect control and future design of anti-insect selective ligands.
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x ray structure and mutagenesis of the scorpion depressant toxin lqhit2 reveals key determinants crucial for activity and anti insect selectivity
Journal of Molecular Biology, 2007Co-Authors: Izhar Karbat, Michel Gurevitz, Lior Cohen, Roy Kahn, Dalia Gordon, Michael Turkov, Felix FrolowAbstract:Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for Receptor Site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the Receptor Site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the Receptor Site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.
