Publications by authors named "Michael Gurevitz"

55 Publications

Ferredoxin-mediated reduction of 2-nitrothiophene inhibits photosynthesis: mechanism and herbicidal potential.

Biochem J 2020 03;477(6):1149-1158

Department of Plant Molecular Biology and Ecology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

Searching for compounds that inhibit the growth of photosynthetic organisms highlighted a prominent effect at micromolar concentrations of the nitroheteroaromatic thioether, 2-nitrothiophene, applied in the light. Since similar effects were reminiscent to those obtained also by radicals produced under excessive illumination or by herbicides, and in light of its redox potential, we suspected that 2-nitrothiophene was reduced by ferredoxin, a major reducing compound in the light. In silico examination using docking and tunneling computing algorithms of the putative interaction between 2-nitrothiophene and cyanobacterial ferredoxin has suggested a site of interaction enabling robust electron transfer from the iron-sulfur cluster of ferredoxin to the nitro group of 2-nitrothiophene. ESR and oximetry analyses of cyanobacterial cells (Anabaena PCC7120) treated with 50 μM 2-nitrothiophene under illumination revealed accumulation of oxygen radicals and peroxides. Gas chromatography mass spectrometry analysis of 2-nitrothiophene-treated cells identified cytotoxic nitroso and non-toxic amino derivatives. These products of the degradation pathway of 2-nitrohiophene, which initializes with a single electron transfer that forms a short-live anion radical, are then decomposed to nitrate and thiophene, and may be further reduced to a nitroso hydroxylamine and amino derivatives. This mechanism of toxicity is similar to that of nitroimidazoles (e.g. ornidazole and metronidazole) reduced by ferredoxin in anaerobic bacteria and protozoa, but differs from that of ornidazole in planta.
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http://dx.doi.org/10.1042/BCJ20190830DOI Listing
March 2020

Pore-modulating toxins exploit inherent slow inactivation to block K channels.

Proc Natl Acad Sci U S A 2019 09 23;116(37):18700-18709. Epub 2019 Aug 23.

Department of Biomolecular Sciences, Weizmann Institute of Science, 76100 Rehovot, Israel;

Voltage-dependent potassium channels (Ks) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K-selective pore. Animal toxins targeting Ks are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of K1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K channels as a novel pharmacological target and provides a rational framework for drug design.
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http://dx.doi.org/10.1073/pnas.1908903116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744907PMC
September 2019

The drug ornidazole inhibits photosynthesis in a different mechanism described for protozoa and anaerobic bacteria.

Biochem J 2016 12 19;473(23):4413-4426. Epub 2016 Sep 19.

Department of Plant Molecular Biology & Ecology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 6997801, Israel.

Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle.
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http://dx.doi.org/10.1042/BCJ20160433DOI Listing
December 2016

The specificity of Av3 sea anemone toxin for arthropods is determined at linker DI/SS2-S6 in the pore module of target sodium channels.

Biochem J 2014 Oct;463(2):271-7

*Department of Plant Molecular Biology & Ecology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

Av3 is a peptide neurotoxin from the sea anemone Anemonia viridis that shows specificity for arthropod voltage-gated sodium channels (Navs). Interestingly, Av3 competes with a scorpion α-toxin on binding to insect Navs and similarly inhibits the inactivation process, and thus has been classified as 'receptor site-3 toxin', although the two peptides are structurally unrelated. This raises questions as to commonalities and differences in the way both toxins interact with Navs. Recently, site-3 was partly resolved for scorpion α-toxins highlighting S1-S2 and S3-S4 external linkers at the DIV voltage-sensor module and the juxtaposed external linkers at the DI pore module. To uncover channel determinants involved in Av3 specificity for arthropods, the toxin was examined on channel chimaeras constructed with the external linkers of the mammalian brain Nav1.2a, which is insensitive to Av3, in the background of the Drosophila DmNav1. This approach highlighted the role of linker DI/SS2-S6, adjacent to the channel pore, in determining Av3 specificity. Point mutagenesis at DI/SS2-S6 accompanied by functional assays highlighted Trp404 and His405 as a putative point of Av3 interaction with DmNav1. His405 conservation in arthropod Navs compared with tyrosine in vertebrate Navs may represent an ancient substitution that explains the contemporary selectivity of Av3. Trp404 and His405 localization near the membrane surface and the hydrophobic bioactive surface of Av3 suggest that the toxin possibly binds at a cleft by DI/S6. A partial overlap in receptor site-3 of both toxins nearby DI/S6 may explain their binding competition capabilities.
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http://dx.doi.org/10.1042/BJ20140576DOI Listing
October 2014

Direct evidence that scorpion α-toxins (site-3) modulate sodium channel inactivation by hindrance of voltage-sensor movements.

PLoS One 2013 26;8(11):e77758. Epub 2013 Nov 26.

Department of Biochemistry and Biophysics, Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3 (+) trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841157PMC
October 2014

Convergent evolution of sodium ion selectivity in metazoan neuronal signaling.

Cell Rep 2012 Aug 26;2(2):242-8. Epub 2012 Jul 26.

Department of Molecular Biology and Ecology of Plants, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel.

Ion selectivity of metazoan voltage-gated Na(+) channels is critical for neuronal signaling and has long been attributed to a ring of four conserved amino acids that constitute the ion selectivity filter (SF) at the channel pore. Yet, in addition to channels with a preference for Ca(2+) ions, the expression and characterization of Na(+) channel homologs from the sea anemone Nematostella vectensis, a member of the early-branching metazoan phylum Cnidaria, revealed a sodium-selective channel bearing a noncanonical SF. Mutagenesis and physiological assays suggest that pore elements additional to the SF determine the preference for Na(+) in this channel. Phylogenetic analysis assigns the Nematostella Na(+)-selective channel to a channel group unique to Cnidaria, which diverged >540 million years ago from Ca(2+)-conducting Na(+) channel homologs. The identification of Cnidarian Na(+)-selective ion channels distinct from the channels of bilaterian animals indicates that selectivity for Na(+) in neuronal signaling emerged independently in these two animal lineages.
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http://dx.doi.org/10.1016/j.celrep.2012.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809514PMC
August 2012

Mapping the interaction site for a β-scorpion toxin in the pore module of domain III of voltage-gated Na(+) channels.

J Biol Chem 2012 Aug 2;287(36):30719-28. Epub 2012 Jul 2.

Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA.

Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for β-scorpion toxins on Na(V) channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.
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http://dx.doi.org/10.1074/jbc.M112.370742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436316PMC
August 2012

Mapping of scorpion toxin receptor sites at voltage-gated sodium channels.

Authors:
Michael Gurevitz

Toxicon 2012 Sep 4;60(4):502-11. Epub 2012 Apr 4.

Department of Plant Molecular Biology & Ecology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain II, while Lqh2 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain IV. Double-mutant cycle analysis and dissociation assays employing a battery of Lqh2 mutants against rNa(v)1.2a mutants identified the docking orientation of alpha toxins at the channel external surface of the Gating-module in domain IV. The other point of interaction between the toxin and the channel has not yet been defined and may involve channel residues of either the Pore-module or the Gating-module.
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http://dx.doi.org/10.1016/j.toxicon.2012.03.022DOI Listing
September 2012

Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones.

Proc Biol Sci 2012 Apr 2;279(1732):1351-8. Epub 2011 Nov 2.

Department of Molecular Biology and Ecology of Plants, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.
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http://dx.doi.org/10.1098/rspb.2011.1731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282367PMC
April 2012

Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor.

Proc Natl Acad Sci U S A 2011 Sep 29;108(37):15426-31. Epub 2011 Aug 29.

Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA 98195-7280, USA.

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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http://dx.doi.org/10.1073/pnas.1112320108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174582PMC
September 2011

Elucidation of the molecular basis of selective recognition uncovers the interaction site for the core domain of scorpion alpha-toxins on sodium channels.

J Biol Chem 2011 Oct 8;286(40):35209-17. Epub 2011 Aug 8.

Department of Plant Molecular Biology and Ecology, George S Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel.

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.
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http://dx.doi.org/10.1074/jbc.M111.259507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186375PMC
October 2011

Structure-function map of the receptor site for β-scorpion toxins in domain II of voltage-gated sodium channels.

J Biol Chem 2011 Sep 27;286(38):33641-51. Epub 2011 Jul 27.

Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.

Voltage-gated sodium (Na(v)) channels are the molecular targets of β-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of β-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.
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http://dx.doi.org/10.1074/jbc.M111.282509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190924PMC
September 2011

Rubisco mutagenesis provides new insight into limitations on photosynthesis and growth in Synechocystis PCC6803.

J Exp Bot 2011 Aug 6;62(12):4173-82. Epub 2011 May 6.

Department of Molecular Biology and Ecology of Plants, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (K(cat)) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40-50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced K(cat) so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (P(max)) under saturating irradiance and Ci concentrations indicate that a >60% reduction in K(cat) can be tolerated before P(max) in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH.
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http://dx.doi.org/10.1093/jxb/err116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153676PMC
August 2011

Substitutions in the domain III voltage-sensing module enhance the sensitivity of an insect sodium channel to a scorpion beta-toxin.

J Biol Chem 2011 May 15;286(18):15781-8. Epub 2011 Mar 15.

Department of Entomology, Michigan State University, East Lansing, Michigan 48824, USA.

Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion β-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.
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http://dx.doi.org/10.1074/jbc.M110.217000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091187PMC
May 2011

Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

J Biol Chem 2010 Oct 3;285(40):30531-8. Epub 2010 Aug 3.

Department of Plant Sciences, George S Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel.

Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.
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http://dx.doi.org/10.1074/jbc.M110.150888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945547PMC
October 2010

Coupling between residues on S4 and S1 defines the voltage-sensor resting conformation in NaChBac.

Biophys J 2010 Jul;99(2):456-63

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.
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http://dx.doi.org/10.1016/j.bpj.2010.04.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905116PMC
July 2010

Positions under positive selection--key for selectivity and potency of scorpion alpha-toxins.

Mol Biol Evol 2010 May 17;27(5):1025-34. Epub 2009 Dec 17.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.
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http://dx.doi.org/10.1093/molbev/msp310DOI Listing
May 2010

Fusion and retrotransposition events in the evolution of the sea anemone Anemonia viridis neurotoxin genes.

J Mol Evol 2009 Aug 16;69(2):115-24. Epub 2009 Jul 16.

Department of Plant Sciences, Tel-Aviv University, Ramat-Aviv, Israel.

Sea anemones are sessile predators that use a variety of toxins to paralyze prey and foe. Among these toxins, Types I, II and III are short peptides that affect voltage-gated sodium channels. Anemonia viridis is the only sea anemone species that produces both Types I and III neurotoxin. Although the two toxin types are unrelated in sequence and three-dimensional structure, cloning and comparative analysis of their loci revealed a highly similar sequence at the 5' region, which encodes a signal peptide. This similarity was likely generated by gene fusion and could be advantageous in transcript stability and intracellular trafficking and secretion. In addition, these analyses identified the processed pseudogenes of the two gene families in the genome of A. viridis, probably resulting from retrotransposition events. As presence of processed pseudogenes in the genome requires transcription in germ-line cells, we analyzed oocyte-rich ovaries and found that indeed they contain Types I and III transcripts. This result raises questions regarding the role of toxin transcripts in these tissues. Overall, the retrotransposition and gene fusion events suggest that the genes of both Types I and III neurotoxins evolved in a similar fashion and share a partial common ancestry.
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http://dx.doi.org/10.1007/s00239-009-9258-xDOI Listing
August 2009

Drosomycin, an innate immunity peptide of Drosophila melanogaster, interacts with the fly voltage-gated sodium channel.

J Biol Chem 2009 Aug 1;284(35):23558-63. Epub 2009 Jul 1.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSalphabeta scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion beta-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion beta-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhalphaIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNa(v)1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNa(v)1 activation. Thus, the short CSalphabeta scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNa(v)1.
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http://dx.doi.org/10.1074/jbc.M109.023358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749130PMC
August 2009

Molecular requirements for recognition of brain voltage-gated sodium channels by scorpion alpha-toxins.

J Biol Chem 2009 Jul 9;284(31):20684-91. Epub 2009 Jun 9.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat-Aviv 69978, Tel Aviv, Israel.

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.
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http://dx.doi.org/10.1074/jbc.M109.021303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742833PMC
July 2009

Sea anemone toxins affecting voltage-gated sodium channels--molecular and evolutionary features.

Toxicon 2009 Dec 5;54(8):1089-101. Epub 2009 Mar 5.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

The venom of sea anemones is rich in low molecular weight proteinaceous neurotoxins that vary greatly in structure, site of action, and phyletic (insect, crustacean or vertebrate) preference. This toxic versatility likely contributes to the ability of these sessile animals to inhabit marine environments co-habited by a variety of mobile predators. Among these toxins, those that show prominent activity at voltage-gated sodium channels and are critical in predation and defense, have been extensively studied for more than three decades. These studies initially focused on the discovery of new toxins, determination of their covalent and folded structures, understanding of their mechanisms of action on different sodium channels, and identification of the primary sites of interaction of the toxins with their channel receptors. The channel binding site for Type I and the structurally unrelated Type III sea anemone toxins was identified as neurotoxin receptor site 3, a site previously shown to be targeted by scorpion alpha-toxins. The bioactive surfaces of toxin representatives from these two sea anemone types have been characterized by mutagenesis. These analyses pointed to heterogeneity of receptor site 3 at various sodium channels. A turning point in evolutionary studies of sea anemone toxins was the recent release of the genome sequence of Nematostella vectensis, which enabled analysis of the genomic organization of the corresponding genes. This analysis demonstrated that Type I toxins in Nematostella and other species are encoded by gene families and suggested that these genes developed by concerted evolution. The current review provides a brief historical description of the discovery and characterization of sea anemone toxins that affect voltage-gated sodium channels and delineates recent advances in the study of their structure-activity relationship and evolution.
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http://dx.doi.org/10.1016/j.toxicon.2009.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807626PMC
December 2009

Intron retention as a posttranscriptional regulatory mechanism of neurotoxin expression at early life stages of the starlet anemone Nematostella vectensis.

J Mol Biol 2008 Jul 11;380(3):437-43. Epub 2008 May 11.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

Sea anemones use an arsenal of peptide neurotoxins accumulated in special stinging cells (nematocytes) for defense and predation. Intriguingly, genomic analysis of Nematostella vectensis revealed only a single toxin, Nv1 (N. vectensis toxin 1), encoded by multiple extremely conserved genes. We examined the toxic potential of Nv1 and whether it is produced by the three developmental stages (embryo, planula, and polyp) of Nematostella. Nv1 was expressed in recombinant form and, similarly to Type I sea anemone toxins, inhibited the inactivation of voltage-gated sodium channels. However, in contrast to the other toxins, Nv1 revealed high specificity for insect over mammalian voltage-gated sodium channels. Transcript analysis indicated that multiple Nv1 loci are transcribed at all developmental stages of N. vectensis, whereas splicing of these transcripts is restricted to the polyp stage. This finding suggests that regulation of Nv1 synthesis is posttranscriptional and that the embryo and planula stages do not produce the Nv1 toxin. This rare phenomenon of intron retention at the early developmental stages is intriguing and raises the question as to the mechanism enabling such differential expression in sea anemones.
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http://dx.doi.org/10.1016/j.jmb.2008.05.011DOI Listing
July 2008

Miniaturization of scorpion beta-toxins uncovers a putative ancestral surface of interaction with voltage-gated sodium channels.

J Biol Chem 2008 May 13;283(22):15169-76. Epub 2008 Mar 13.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel.

The bioactive surface of scorpion beta-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved betaalphabetabeta core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian beta-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated beta-toxins (DeltaDeltaBj-xtrIT and DeltaDeltaCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, DeltaDeltaBj-xtrIT and DeltaDeltaCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion alpha-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion alpha-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhalphaIT at insect sodium channels. Co-application of DeltaDeltaBj-xtrIT or DeltaDeltaCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at DeltaDeltaBj-xtrIT and DeltaDeltaCss4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both beta-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.
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http://dx.doi.org/10.1074/jbc.M801229200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397468PMC
May 2008

Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome.

Mol Biol Evol 2008 Apr 24;25(4):737-47. Epub 2008 Jan 24.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.

Gene families, which encode toxins, are found in many poisonous animals, yet there is limited understanding of their evolution at the nucleotide level. The release of the genome draft sequence for the sea anemone Nematostella vectensis enabled a comprehensive study of a gene family whose neurotoxin products affect voltage-gated sodium channels. All gene family members are clustered in a highly repetitive approximately 30-kb genomic region and encode a single toxin, Nv1. These genes exhibit extreme conservation at the nucleotide level which cannot be explained by purifying selection. This conservation greatly differs from the toxin gene families of other animals (e.g., snakes, scorpions, and cone snails), whose evolution was driven by diversifying selection, thereby generating a high degree of genetic diversity. The low nucleotide diversity at the Nv1 genes is reminiscent of that reported for DNA encoding ribosomal RNA (rDNA) and 2 hsp70 genes from Drosophila, which have evolved via concerted evolution. This evolutionary pattern was experimentally demonstrated in yeast rDNA and was shown to involve unequal crossing-over. Through sequence analysis of toxin genes from multiple N. vectensis populations and 2 other anemone species, Anemonia viridis and Actinia equina, we observed that the toxin genes for each sea anemone species are more similar to one another than to those of other species, suggesting they evolved by manner of concerted evolution. Furthermore, in 2 of the species (A. viridis and A. equina) we found genes that evolved under diversifying selection, suggesting that concerted evolution and accelerated evolution may occur simultaneously.
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http://dx.doi.org/10.1093/molbev/msn021DOI Listing
April 2008

NMR analysis of interaction of LqhalphaIT scorpion toxin with a peptide corresponding to the D4/S3-S4 loop of insect para voltage-gated sodium channel.

Biochemistry 2008 Jan 23;47(3):911-21. Epub 2007 Dec 23.

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.
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http://dx.doi.org/10.1021/bi701323kDOI Listing
January 2008

Mammalian skeletal muscle voltage-gated sodium channels are affected by scorpion depressant "insect-selective" toxins when preconditioned.

Mol Pharmacol 2007 Nov 24;72(5):1220-7. Epub 2007 Aug 24.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.

Among scorpion beta- and alpha-toxins that modify the activation and inactivation of voltage-gated sodium channels (Na(v)s), depressant beta-toxins have traditionally been classified as anti-insect selective on the basis of toxicity assays and lack of binding and effect on mammalian Na(v)s. Here we show that the depressant beta-toxins LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus (Lqh) bind with nanomolar affinity to receptor site 4 on rat skeletal muscle Na(v)s, but their effect on the gating properties can be viewed only after channel preconditioning, such as that rendered by a long depolarizing prepulse. This observation explains the lack of toxicity when depressant toxins are injected in mice. However, when the muscle channel rNa(v)1.4, expressed in Xenopus laevis oocytes, was modulated by the site 3 alpha-toxin LqhalphaIT, LqhIT2 was capable of inducing a negative shift in the voltage-dependence of activation after a short prepulse, as was shown for other beta-toxins. These unprecedented results suggest that depressant toxins may have a toxic impact on mammals in the context of the complete scorpion venom. To assess whether LqhIT2 and Lqh-dprIT3 interact with the insect and rat muscle channels in a similar manner, we examined the role of Glu24, a conserved "hot spot" at the bioactive surface of beta-toxins. Whereas substitutions E24A/N abolished the activity of both LqhIT2 and Lqh-dprIT3 at insect Na(v)s, they increased the affinity of the toxins for rat skeletal muscle channels. This result implies that depressant toxins interact differently with the two channel types and that substitution of Glu24 is essential for converting toxin selectivity.
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http://dx.doi.org/10.1124/mol.107.039057DOI Listing
November 2007

Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture.

J Biol Chem 2007 Oct 8;282(40):29424-30. Epub 2007 Aug 8.

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, 69978 Tel-Aviv, Israel.

Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.
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http://dx.doi.org/10.1074/jbc.M704651200DOI Listing
October 2007

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.

Biochem J 2007 Aug;406(1):41-8

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.

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 Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-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 Na(v)s, 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 LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s 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 beta-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 DmNa(v)1, 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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http://dx.doi.org/10.1042/BJ20070233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1948988PMC
August 2007

The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail.

FEBS J 2007 Apr 9;274(8):1918-31. Epub 2007 Mar 9.

Department of Plant Sciences, George S.Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05737.xDOI Listing
April 2007