The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
William A. Catterall - One of the best experts on this subject based on the ideXlab platform.
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mapping the receptor sites for a β Scorpion Toxin on the pore module in domain iii of voltage gated sodium channels
Biophysical Journal, 2012Co-Authors: Joel Z Zhang, Michel Gurevitz, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, Vladimir Yarovyarovoy, William A. CatterallAbstract:Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. The -Scorpion Toxin CssIV traps a voltage-sensor of Nav channels in its activated state via a voltage-sensor trapping mechanism and thus shifts their voltage dependence of activation to more negative membrane potentials. The SS2-S6 linker of the pore domain in domain III (IIISS2-S6) is crucial in determining the action of CssIV upon Nav channels. We found that five substitutions at four amino acid residues in IIISS2-S6 markedly alter voltage-sensor trapping current (IVST) by a recombinant Toxin derivative, CssIVE15A. These residues are concentrated in the region between N1436 and D1445 and form a discontinuous interaction site. Three of them (E1438A, D1445A and D1445Y) markedly decrease IVST, whereas the other two (N1436G and L1439A) markedly increase IVST. N1436G increases binding affinity of CssIVE15A to Nav channels in the resting state, whereas L1439A increases the efficacy of trapping the activated voltage-sensor by the prebound Toxin. Time courses of voltage sensor trapping for the WT and mutant channels fit an allosteric kinetic model that includes a lower affinity resting state and a higher affinity activated/trapped state. Structural modeling suggests that the IIISS2-S6 is in close proximity to the IIS1-S2 and IIS3-S4 linkers in 3D space and that the bound Toxin sits in a cleft formed by these three extracellular loops. Our results define the molecular map of a third interacting component of the -Scorpion Toxin receptor site of mammalian Nav channels and provide new molecular details of the voltage-sensor trapping mechanism of Toxin action.
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Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Biophysical Journal, 2010Co-Authors: Zhongli Zhang, Michel Gurevitz, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, William A. CatterallAbstract: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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Structure and function of the voltage sensor of sodium channels probed by a beta-Scorpion Toxin.
Journal of Biological Chemistry, 2006Co-Authors: Sandrine Cestèle, Todd Scheuer, Vladimir Yarov-yarovoy, François Sampieri, William A. CatterallAbstract:Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion Toxins bind specifically to neuroToxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-Scorpion Toxin binds to the resting state, and subsequently the bound Toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-Scorpion Toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-Scorpion Toxin binding but alter voltage dependence of activation and enhance beta-Scorpion Toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the Toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of Scorpion Toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
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Structure and function of the voltage sensor of sodium channels probed by a β-Scorpion Toxin
Journal of Biological Chemistry, 2006Co-Authors: Sandrine Cestèle, Todd Scheuer, Vladimir Yarov-yarovoy, François Sampieri, William A. CatterallAbstract:Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion Toxins bind specifically to neuroToxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-Scorpion Toxin binds to the resting state, and subsequently the bound Toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-Scorpion Toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-Scorpion Toxin binding but alter voltage dependence of activation and enhance beta-Scorpion Toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the Toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of Scorpion Toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
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Neutralization of gating charges in domain II of the sodium channel α subunit enhances voltage-sensor trapping by a β-Scorpion Toxin
Journal of General Physiology, 2001Co-Authors: Sandrine Cestèle, Todd Scheuer, Massimo Mantegazza, William A. CatterallAbstract:β-Scorpion Toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-Scorpion Toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-Scorpion Toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-Scorpion Toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound Toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-Scorpion Toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances Toxin-induced channel activation.
Jinghai Zhang - One of the best experts on this subject based on the ideXlab platform.
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The roles of conserved aromatic residues (Tyr5 and Tyr42) in interaction of Scorpion Toxin BmK AGP-SYPU1 with human Nav1.7.
International Journal of Biological Macromolecules, 2017Co-Authors: Xiangxue Meng, Mingyi Zhao, Fangyang Wang, Yao Jin, Yanfeng Liu, Yongbo Song, Xue Hou, Jinghai ZhangAbstract:Scorpion Toxins are invaluable source of therapeutic leads and pharmacological tools which produce influence on the voltage gated sodium channels. In the previous study, our group has reported BmK AGP-SYPU1 (64 amino acids), one Scorpion Toxin with both potential α-type and β-type Scorpion characteristics and analgesic activity in vivo, act as an activator to hNav1.4 and hNav1.5. Additionally, conserved aromatic amino acids Tyr5 and Tyr42 played important roles in bioactivity of BmK AGP-SYPU1 on hNav1.4 and hNav1.5. However, the anti-nonceptitor mechanisms of BmK AGP-SYPU1 referred in vivo have not been clarified yet. The roles of Tyr5 and Tyr42 have still kept unclear in the interaction of BmK AGP-SYPU1 with other VGSCs. In this study, in order to give a deep insight into the relationship of structure and function, the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.7, which has emerged as a key molecule involved in pain processing, were examined by using Na+-specialized fluorescent dye and the whole-cell patch clamp. The data showed that BmK AGP-SYPU1 also displayed as an activator to hNav1.7 with the potential characteristic of α-type and β-type Scorpion Toxin. Both Tyr5 and Tyr42 were the key amino acids to the functional properties of BmK AGP-SYPU1 when interacting with hNav1.7, and the Tyr42 was involved in the preferences of the Toxin to distinct action sites of hNav1.7. On the whole, these data provided more information about the structure of BmK AGP-SYPU1 related to its function upon hNav1.7, and may bring some clues for engineering Scorpion Toxins to achieve high bioactivity with lower side effects.
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The Functional Property Changes of Muscular Nav1.4 and Cardiac Nav1.5 Induced by Scorpion Toxin BmK AGP-SYPU1 Mutants Y42F and Y5F
Biochemistry, 2015Co-Authors: Xiangxue Meng, Mingyi Zhao, Fangyang Wang, Yao Jin, Yanfeng Liu, Yongbo Song, Jinghai ZhangAbstract:Scorpion Toxins are invaluable therapeutic leads and pharmacological tools which influence the voltage-gated sodium channels. However, the details were still unclear about the structure–function relationship of Scorpion Toxins on VGSC subtypes. In the previous study, we reported one α-type Scorpion Toxin Bmk AGP-SYPU1 and its two mutants (Y5F and Y42F) which had been demonstrated to ease pain in mice acetic acid writhing test. However, the function of Bmk AGP-SYPU1 on VGSCs is still unknown. In this study, we examined the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.4 and hNav1.5 heterologously expressed CHO cell lines by using Na+-specialized fluorescent dye and whole-cell patch clamp. The data showed that BmK AGP-SYPU1 displayed as an activator of hNav1.4 and hNav1.5, which might indeed contribute to its biotoxicity to muscular and cardiac system and exhibited the functional properties of both the α-type and β-type Scorpion Toxin. Notably, Y5F mutant exhibited lower activatory ef...
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Soluble expression, purification and the role of C-terminal glycine residues in Scorpion Toxin BmK AGP-SYPU2.
BMB reports, 2010Co-Authors: Rong Zhang, Yongbo Song, Yong Cui, Xi Zhang, Zhuo Yang, Yong Shan Zhao, Jinghai ZhangAbstract:The existence of glycine residues in long-chain Scorpion Toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-Scorpion Toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.
Todd Scheuer - One of the best experts on this subject based on the ideXlab platform.
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mapping the receptor sites for a β Scorpion Toxin on the pore module in domain iii of voltage gated sodium channels
Biophysical Journal, 2012Co-Authors: Joel Z Zhang, Michel Gurevitz, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, Vladimir Yarovyarovoy, William A. CatterallAbstract:Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. The -Scorpion Toxin CssIV traps a voltage-sensor of Nav channels in its activated state via a voltage-sensor trapping mechanism and thus shifts their voltage dependence of activation to more negative membrane potentials. The SS2-S6 linker of the pore domain in domain III (IIISS2-S6) is crucial in determining the action of CssIV upon Nav channels. We found that five substitutions at four amino acid residues in IIISS2-S6 markedly alter voltage-sensor trapping current (IVST) by a recombinant Toxin derivative, CssIVE15A. These residues are concentrated in the region between N1436 and D1445 and form a discontinuous interaction site. Three of them (E1438A, D1445A and D1445Y) markedly decrease IVST, whereas the other two (N1436G and L1439A) markedly increase IVST. N1436G increases binding affinity of CssIVE15A to Nav channels in the resting state, whereas L1439A increases the efficacy of trapping the activated voltage-sensor by the prebound Toxin. Time courses of voltage sensor trapping for the WT and mutant channels fit an allosteric kinetic model that includes a lower affinity resting state and a higher affinity activated/trapped state. Structural modeling suggests that the IIISS2-S6 is in close proximity to the IIS1-S2 and IIS3-S4 linkers in 3D space and that the bound Toxin sits in a cleft formed by these three extracellular loops. Our results define the molecular map of a third interacting component of the -Scorpion Toxin receptor site of mammalian Nav channels and provide new molecular details of the voltage-sensor trapping mechanism of Toxin action.
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Mapping the β-Scorpion Toxin Receptor Site on Voltage-Gated Sodium Channels
Biophysical Journal, 2010Co-Authors: Zhongli Zhang, Michel Gurevitz, Lior Cohen, Izhar Karbat, Dalia Gordon, Todd Scheuer, William A. CatterallAbstract: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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Structure and function of the voltage sensor of sodium channels probed by a beta-Scorpion Toxin.
Journal of Biological Chemistry, 2006Co-Authors: Sandrine Cestèle, Todd Scheuer, Vladimir Yarov-yarovoy, François Sampieri, William A. CatterallAbstract:Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion Toxins bind specifically to neuroToxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-Scorpion Toxin binds to the resting state, and subsequently the bound Toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-Scorpion Toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-Scorpion Toxin binding but alter voltage dependence of activation and enhance beta-Scorpion Toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the Toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of Scorpion Toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
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Structure and function of the voltage sensor of sodium channels probed by a β-Scorpion Toxin
Journal of Biological Chemistry, 2006Co-Authors: Sandrine Cestèle, Todd Scheuer, Vladimir Yarov-yarovoy, François Sampieri, William A. CatterallAbstract:Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion Toxins bind specifically to neuroToxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-Scorpion Toxin binds to the resting state, and subsequently the bound Toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-Scorpion Toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-Scorpion Toxin binding but alter voltage dependence of activation and enhance beta-Scorpion Toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the Toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of Scorpion Toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
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Neutralization of gating charges in domain II of the sodium channel α subunit enhances voltage-sensor trapping by a β-Scorpion Toxin
Journal of General Physiology, 2001Co-Authors: Sandrine Cestèle, Todd Scheuer, Massimo Mantegazza, William A. CatterallAbstract:β-Scorpion Toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-Scorpion Toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-Scorpion Toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-Scorpion Toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound Toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-Scorpion Toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances Toxin-induced channel activation.
Sandrine Cestèle - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of the voltage sensor of sodium channels probed by a beta-Scorpion Toxin.
Journal of Biological Chemistry, 2006Co-Authors: Sandrine Cestèle, Todd Scheuer, Vladimir Yarov-yarovoy, François Sampieri, William A. CatterallAbstract:Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion Toxins bind specifically to neuroToxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-Scorpion Toxin binds to the resting state, and subsequently the bound Toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-Scorpion Toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-Scorpion Toxin binding but alter voltage dependence of activation and enhance beta-Scorpion Toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the Toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of Scorpion Toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
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Structure and function of the voltage sensor of sodium channels probed by a β-Scorpion Toxin
Journal of Biological Chemistry, 2006Co-Authors: Sandrine Cestèle, Todd Scheuer, Vladimir Yarov-yarovoy, François Sampieri, William A. CatterallAbstract:Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion Toxins bind specifically to neuroToxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-Scorpion Toxin binds to the resting state, and subsequently the bound Toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-Scorpion Toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-Scorpion Toxin binding but alter voltage dependence of activation and enhance beta-Scorpion Toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the Toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of Scorpion Toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.
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Molecular cloning and functional expression of the alpha-Scorpion Toxin BotIII: pivotal role of the C-terminal region for its interaction with voltage-dependent sodium channels.
Peptides, 2004Co-Authors: Khadija Benkhadir, Mohamed El Ayeb, Riadh Kharrat, Sandrine Cestèle, Amor Mosbah, Habib KarouiAbstract:Alpha Scorpion Toxins bind to receptor site 3 on voltage-dependent sodium channels and inhibit their inactivation. The alpha-Scorpion Toxin BotIII is the most toxic protein of Buthus occitanus tunetanus. Its sequence differs only by three amino acid residues from that of AahII, the most active alpha-Toxin. Due to their high affinity and selectivity for mammalian sodium channels, BotIII and AahII represent powerful tools for studying the molecular determinants of specificity for voltage-dependent sodium channels. Sequence analysis of BotIII gene has revealed two exons separated by a 381-bp intron and a signal peptide of 19 amino acids. We succeeded in expressing BotIII in significantly higher amounts than AahII the only expressed strict alpha anti-mammalian Scorpion Toxin reported in the literature. We have also modified specific amino acid residues of BotIII. The recombinant and the natural Toxins differ by the amidation of the C-terminal residue. Toxicity and binding experiments indicated: (a) the affinity of rBotIII-OH and rAahII-OH (rBotIII-OH with the 3 mutations R10V, V51L, N64H) for the voltage-dependent sodium channels is reduced compared to the natural Toxins. This data revealed the important role of the C-terminal amidation for the biological activity of BotIII and AahII; (b) the single mutation N64H is responsible for the difference of toxicity and affinity between rBotIII-OH and rAahII-OH; (c) the addition of the sequence GR to rBotIII-OH leads to the loss of biological activity. This study is in agreement with the important role attributed to the C-terminal sequence of alpha-Toxins in their interaction with sodium channels receptors.
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Neutralization of gating charges in domain II of the sodium channel α subunit enhances voltage-sensor trapping by a β-Scorpion Toxin
Journal of General Physiology, 2001Co-Authors: Sandrine Cestèle, Todd Scheuer, Massimo Mantegazza, William A. CatterallAbstract:β-Scorpion Toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-Scorpion Toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-Scorpion Toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-Scorpion Toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound Toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-Scorpion Toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances Toxin-induced channel activation.
Xiangxue Meng - One of the best experts on this subject based on the ideXlab platform.
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The roles of conserved aromatic residues (Tyr5 and Tyr42) in interaction of Scorpion Toxin BmK AGP-SYPU1 with human Nav1.7.
International Journal of Biological Macromolecules, 2017Co-Authors: Xiangxue Meng, Mingyi Zhao, Fangyang Wang, Yao Jin, Yanfeng Liu, Yongbo Song, Xue Hou, Jinghai ZhangAbstract:Scorpion Toxins are invaluable source of therapeutic leads and pharmacological tools which produce influence on the voltage gated sodium channels. In the previous study, our group has reported BmK AGP-SYPU1 (64 amino acids), one Scorpion Toxin with both potential α-type and β-type Scorpion characteristics and analgesic activity in vivo, act as an activator to hNav1.4 and hNav1.5. Additionally, conserved aromatic amino acids Tyr5 and Tyr42 played important roles in bioactivity of BmK AGP-SYPU1 on hNav1.4 and hNav1.5. However, the anti-nonceptitor mechanisms of BmK AGP-SYPU1 referred in vivo have not been clarified yet. The roles of Tyr5 and Tyr42 have still kept unclear in the interaction of BmK AGP-SYPU1 with other VGSCs. In this study, in order to give a deep insight into the relationship of structure and function, the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.7, which has emerged as a key molecule involved in pain processing, were examined by using Na+-specialized fluorescent dye and the whole-cell patch clamp. The data showed that BmK AGP-SYPU1 also displayed as an activator to hNav1.7 with the potential characteristic of α-type and β-type Scorpion Toxin. Both Tyr5 and Tyr42 were the key amino acids to the functional properties of BmK AGP-SYPU1 when interacting with hNav1.7, and the Tyr42 was involved in the preferences of the Toxin to distinct action sites of hNav1.7. On the whole, these data provided more information about the structure of BmK AGP-SYPU1 related to its function upon hNav1.7, and may bring some clues for engineering Scorpion Toxins to achieve high bioactivity with lower side effects.
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The Functional Property Changes of Muscular Nav1.4 and Cardiac Nav1.5 Induced by Scorpion Toxin BmK AGP-SYPU1 Mutants Y42F and Y5F
Biochemistry, 2015Co-Authors: Xiangxue Meng, Mingyi Zhao, Fangyang Wang, Yao Jin, Yanfeng Liu, Yongbo Song, Jinghai ZhangAbstract:Scorpion Toxins are invaluable therapeutic leads and pharmacological tools which influence the voltage-gated sodium channels. However, the details were still unclear about the structure–function relationship of Scorpion Toxins on VGSC subtypes. In the previous study, we reported one α-type Scorpion Toxin Bmk AGP-SYPU1 and its two mutants (Y5F and Y42F) which had been demonstrated to ease pain in mice acetic acid writhing test. However, the function of Bmk AGP-SYPU1 on VGSCs is still unknown. In this study, we examined the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.4 and hNav1.5 heterologously expressed CHO cell lines by using Na+-specialized fluorescent dye and whole-cell patch clamp. The data showed that BmK AGP-SYPU1 displayed as an activator of hNav1.4 and hNav1.5, which might indeed contribute to its biotoxicity to muscular and cardiac system and exhibited the functional properties of both the α-type and β-type Scorpion Toxin. Notably, Y5F mutant exhibited lower activatory ef...