Scanning Mutagenesis of ω-Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker ω-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro10, Asn27, and Arg35, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled ω-atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 μm, ω-atracotoxin-Hv1a had no effect on heterologously expressed rat Cav2.1, Cav2.2, and Cav1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 μm ω-atracotoxin-Hv1a inhibited rat Cav currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn27 and Arg35, which appears to be critical for interaction of the toxin with insect Cav channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro10, Asn27, and Arg35 confers specific binding to insect Cav channels and is largely responsible for the remarkable phyletic selectivity of ω-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Cav channels. We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker ω-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro10, Asn27, and Arg35, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled ω-atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 μm, ω-atracotoxin-Hv1a had no effect on heterologously expressed rat Cav2.1, Cav2.2, and Cav1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 μm ω-atracotoxin-Hv1a inhibited rat Cav currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn27 and Arg35, which appears to be critical for interaction of the toxin with insect Cav channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro10, Asn27, and Arg35 confers specific binding to insect Cav channels and is largely responsible for the remarkable phyletic selectivity of ω-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Cav channels. The first peptide neurotoxins isolated from the venom of Australian funnel-web spiders (genera Atrax and Hadronyche) and shown to have selective activity against insects were members of the ω-atracotoxin-1 (ACTX) 1The abbreviations used are: ACTX, atracotoxin; HVA, high voltage-activated; WT, wild-type; HEK, human embryonic kidney; VGCC, voltage-gated calcium channel; ΔΔGbind, change in the free energy of binding. family (1, Atkinson, R. K., Howden, M. E. H., Tyler, M. I., and Vonarx, E. J. (June 9, 1998) U. S. Patent 5,763,568Google Scholar, 2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar, 3Wang X.-H. Smith R. Fletcher J.I. Wilson H. Wood C.J. Howden M.E.H. King G.F. Eur. J. Biochem. 1999; 264: 488-494Crossref PubMed Scopus (76) Google Scholar). These toxins comprise 36-37 residues with six strictly conserved cysteine residues that are paired to form three disulfide bridges (4Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google Scholar). The best studied family member, ω-ACTX-Hv1a, is one of the most potent insecticidal peptide toxins discovered so far (4Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google Scholar, 5Bloomquist J.R. Invertebr. Neurosci. 2003; 5: 45-50Crossref PubMed Scopus (42) Google Scholar); it has proved lethal to all insect orders that have been tested, including coleopterans, dictyopterans, hemipterans, orthopterans, and refractory lepidopteran pests such as the tobacco budworm Heliothis virescens and the cotton bollworm Helicoverpa armigera (1, Atkinson, R. K., Howden, M. E. H., Tyler, M. I., and Vonarx, E. J. (June 9, 1998) U. S. Patent 5,763,568Google Scholar, 2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar, 5Bloomquist J.R. Invertebr. Neurosci. 2003; 5: 45-50Crossref PubMed Scopus (42) Google Scholar). The phyletic specificity of these toxins appears to reside in their ability to block insect, but not vertebrate, voltage-gated calcium channels (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar). Although nanomolar concentrations of ω-ACTX-Hv1a are sufficient to block high voltage-activated (HVA) calcium currents in the central nervous system of the fruit fly Drosophila melanogaster and the American cockroach Periplaneta americana (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar, 5Bloomquist J.R. Invertebr. Neurosci. 2003; 5: 45-50Crossref PubMed Scopus (42) Google Scholar), 1 μm toxin does not block HVA calcium currents in vertebrate neurons (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar) and the toxin is harmless when injected subcutaneously into newborn mice (1, Atkinson, R. K., Howden, M. E. H., Tyler, M. I., and Vonarx, E. J. (June 9, 1998) U. S. Patent 5,763,568Google Scholar). With the exception of the organophosphate and carbamate insecticides (which target acetylcholinesterase), the vast majority of synthetic insecticides are directed against voltage-gated sodium channels, nicotinic acetylcholine receptors, or GABA/glutamate-gated chloride channels. This limited group of targets has promoted the evolution of cross-resistance to different families of insecticides (6Brogdon W.G. McAllister J.C. Emerg. Infect. Dis. 1998; 4: 605-613Crossref PubMed Scopus (330) Google Scholar) and stimulated interest in the development of new insecticides that act on targets outside of this established ion channel triumvirate (7Maggio F. Sollod B.L. Tedford H.W. King G.F. Gilbert L.I. Iatrou K. Gill S. Comprehensive Molecular Insect Science. Elsevier, 2004Google Scholar). Although blockers of HVA calcium currents have evolved independently in the venoms of cone snails, snakes, and spiders (4Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google Scholar, 8Olivera B.M. Miljanich G.P. Ramachandran J. Adams M.E. Annu. Rev. Biochem. 1994; 63: 823-867Crossref PubMed Scopus (696) Google Scholar, 9de Weille J.R. Schweitz H. Maes P. Tartar A. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2437-2440Crossref PubMed Scopus (211) Google Scholar), no commercially available insecticide exploits this target (7Maggio F. Sollod B.L. Tedford H.W. King G.F. Gilbert L.I. Iatrou K. Gill S. Comprehensive Molecular Insect Science. Elsevier, 2004Google Scholar, 10King G.F. Tedford H.W. Maggio F. J. Toxicol. Toxin Rev. 2002; 21: 359-389Crossref Google Scholar). Thus, ω-ACTX-Hv1a is a valuable lead for the development of a new class of insecticides that target insect calcium channels. The three-dimensional structure of ω-ACTX-Hv1a comprises a disulfide-rich globular core (residues 4-21), with residues 22-37 forming a finger-like β hairpin that protrudes from this globular region (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar). In a previous structure-activity relationship study, we demonstrated the functional significance of residues in the β hairpin H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). In the study, we of ω-ACTX-Hv1a in we a complete panel of alanine all of the toxin we these using assays in the and the by binding assays using a neuronal from the of P. the interaction that is not by of The that a spatially contiguous epitope of residues Pro10, Asn27, and Arg35 is responsible for the specific interaction of this toxin with insect calcium channels. panel of mutants was used to the chemical of these residues that are critical for the This the rational design of chemical insecticides on the structure of of ω-ACTX-Hv1a were into a synthetic ω-ACTX-Hv1a as previously H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar) using using the a of the as a or using a of in with the and as a The of the was with and and into the as previously for H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). were by a in which a of one of the was used as the and of ω-ACTX-Hv1a and mutants was as previously using by high H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). The of toxin was confirmed using Insect activity was as previously H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar, F. King G.F. Toxicon. 2002; PubMed Scopus Google Scholar) by in insect M. E. R. A. M. E. PubMed Scopus Google Scholar) into crickets (Acheta domestica) or flies (Musca domestica). the that is lethal to of were as previously H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar), and the reported are the of to three of ω-ACTX-Hv1a or mutants were and as previously using a H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). The toxin was of assays with crickets that of to of ω-ACTX-Hv1a was not this as to or toxin activity is not a target for the alanine of the region of ω-ACTX-Hv1a H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar) that was we to a in which was mutated to and was mutated to for as a The mutant was functional in assays and it a that to be to to of the mutant was as previously for P. H. R. F. P. A. J. F. PubMed Scopus Google Scholar) for in the high used to the radiolabeled toxin. The was using a μm 1 using a of and in in in by a of in by a of in toxin was to the of the toxin was to the radiolabeled mutant be to as of membranes were prepared from the heads of P. americana as previously P. M. M. J. Biol. 1999; PubMed Scopus Google Scholar), that the was The of in the was using a with as a Competitive using binding were using concentrations of mutant toxin in the of a of binding were to a of in binding and neuronal membranes were to binding to a of of was to a of were for the were by with of and toxin was from the using in The were three with of toxin binding was in the of 10 ω-ACTX-Hv1a and of binding. of the were using to to a for were using the (which a is the of radiolabeled toxin and is Biochem. PubMed Scopus Google Scholar). The used for these was by using the to on from binding with and embryonic were and with and β of rat HVA calcium channels as previously J. Neurosci. 21: PubMed Google Scholar). were from the as previously J. Neurosci. 21: PubMed Google Scholar). of a of previously alanine mutants of ω-ACTX-Hv1a H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar) to a panel of mutants that alanine all to of the toxin. The six cysteine residues that form the (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar, 10King G.F. Tedford H.W. Maggio F. J. Toxicol. Toxin Rev. 2002; 21: 359-389Crossref Google Scholar) were from the their are in disulfide that are critical to the three-dimensional structure of the toxin. was it of the core of the toxin (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar) and is to be critical for toxin and were are in that are to be (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar). with a the residues are to be The and mutant toxins were to and and were not used in the fly of the that we into ω-ACTX-Hv1a were to structural it was to this We this by of the and mutant The of toxin a β with and and H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar); this is consistent with a β hairpin (residues the structure of ω-ACTX-Hv1a (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar) The is presumably by the of this is in of and mutants but in the of a mutant We previously that of the mutants in the region of the toxin were for H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). the of of the constructed alanine mutants in the disulfide-rich globular of the toxin on the of the as for the and mutants in the of the and mutants from that of the toxin. The of the mutant was to that of the toxin that the was as We that the of the mutant is to that of the toxin. We were to that the was in the mutant This that the of this mutant is in a that the chemical of the a formed by three β (residues and (2Fletcher J.I. Smith R. O'Donoghue S.I. Nilges M. Connor M. Howden M.E.H. Christie M.J. King G.F. Nat. Struct. Biol. 1997; 4: 559-566Crossref PubMed Scopus (160) Google Scholar). is the of the and are by far the most residues Sci. 1994; PubMed Scopus Google Scholar). In alanine is one of the residues this in β Sci. 1994; PubMed Scopus Google Scholar). Thus, this β the of β in the system and the of the We that the in for the mutant is the of a of the toxin structure in the region of the (residues for all alanine we the that functional are by structural insecticidal of mutant was by in a with that of toxin on the previously H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar), we mutated residues to be the of the mutant toxin was that of the was that of or We were to on these that and were identified as residues for activity against crickets in to the residues Asn27 and Arg35 that were identified in previous structure-activity relationship H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). and were identified as for the assays to a for are the most insects from a human and responsible for the of and (4Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google Scholar, F. Sollod B.L. Tedford H.W. King G.F. Gilbert L.I. Iatrou K. Gill S. Comprehensive Molecular Insect Science. Elsevier, 2004Google Scholar). Thus, it was of interest to ω-ACTX-Hv1a as a lead for development of insecticides directed against of human such as cone are spiders are that on a of and Thus, be selective for spiders that evolved venoms of with in ion channel targets different in to the by the high of and of channel R. Science. 2003; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). to this to have been that a of toxins B.L. Wilson R. King G.F. 2004; Scholar). is that a toxin both a core that is for interaction with the target residues that are critical to channel binding in insect but not this we to the epitope of ω-ACTX-Hv1a was insect orders and or residues be for activity in one of these orders but not the we were that the assays to that the epitope of ω-ACTX-Hv1a comprises a residues, the that in toxin be in activity of the mutant in the the activity of a toxin not on for the target channel but on in and ability to The is in that ω-ACTX-Hv1a targets the central nervous system of insects (4Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google Scholar, 5Bloomquist J.R. Invertebr. Neurosci. 2003; 5: 45-50Crossref PubMed Scopus (42) Google Scholar), most toxins act insect Thus, a mutant toxin have activity not of a for the target but of to or limited to the central nervous We that these be critical in the flies as crickets to toxin that are and were to the toxin crickets the insecticidal of alanine mutant in flies to the activity of the toxin. for the residues Pro10, Asn27, and Arg35 were to be a in In and all residues identified as in the assays and for activity against toxin H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar) that in of the three residues most critical for insecticidal activity had a in flies toxin not Competitive a binding to the interaction ω-ACTX-Hv1a and calcium channel target in a not by of and This the ability of ω-ACTX-Hv1a and alanine mutants to the radiolabeled from neuronal membranes prepared from the heads of The was by to be high and binding of to the a that not be of the using or toxin binding or binding We this from the low of ω-ACTX-Hv1a binding consistent with the low of voltage-gated calcium channels in membranes of Scholar); the low of the In with ω-ACTX-Hv1a, the toxin to cockroach neuronal membranes with and R. M. J. Biol. 2004; PubMed Scopus Google Scholar). The low and low of ω-ACTX-Hv1a for these the of of for binding and a high of in most we binding that not by and which be to to for of binding that the of the toxin for cockroach neuronal membranes was Although the of for cockroach neuronal the is to that for the toxin P. M. M. J. Biol. 1999; PubMed Scopus Google Scholar). The was used with the to the in for mutant to alanine of of the three residues critical for against both crickets and flies Pro10, Asn27, and the by with the a in binding The which activity against crickets but not a in as the the binding of ω-ACTX-Hv1a to cockroach neuronal membranes. Thus, as in a previous of neurotoxins E. S. M. J. Biol. 1997; PubMed Scopus Google Scholar), to be a the of the binding and of mutants does not to be a in the for the which was in binding in of the Toxin alanine that Pro10, Asn27, and Arg35 are the of the toxin and are of importance in and but in using a panel of we previously that the group of Asn27 most a to on the target calcium the group of Arg35 most ion with a group on the channel H.W. Fletcher J.I. King G.F. J. Biol. PubMed Scopus Google Scholar). We to the chemical of and to the of their interaction with calcium channels. of toxins with and not that their were to that of toxin. mutant was with toxin in the which that the group is and that this the channel the with this of with a a mutant toxin that had to that of the and In of with a alanine or a a in We that with a in the channel that be by or this mutant was to crickets a mutant and had a toxin is to from these the of the of a or a of these is responsible for the in activity by the The that the which is in the does not critical with the target in was a in We these to that the of with on the channel and that this interaction be to when is by the in which the is of ω-ACTX-Hv1a on the effect of ω-ACTX-Hv1a and a toxin on calcium currents from that expressed rat Cav2.1, Cav2.2, or Cav1.2 calcium channels. At a of 10 μm, ω-ACTX-Hv1a the toxin had effect on calcium currents by these channels not a of 30 μm, the toxin all three channel the inhibition was against Cav1.2 channels the inhibition of and currents was in the the block was for the Cav1.2 The that the block this high toxin is not by the that the inhibition of rat HVA calcium channels on structural that are different from in block of insect calcium channels. as calcium channel blockers have been isolated from the venoms of including from from the families of from the American funnel-web and families of from of Australian funnel-web spiders (genera Atrax and Hadronyche) (7Maggio F. Sollod B.L. Tedford H.W. King G.F. Gilbert L.I. Iatrou K. Gill S. Comprehensive Molecular Insect Science. Elsevier, 2004Google Scholar). The evolution of calcium channel blockers in venoms as of a for is not voltage-gated calcium channels critical in in The of insect is by the that the melanogaster appears to for one family and one family PubMed Scopus Google Scholar), and in of these is embryonic lethal 1998; PubMed Google Scholar, A. J.C. 1998; Google Scholar, F. J. Neurosci. 2002; PubMed Google Scholar). the that insect to be insecticide as first a M. M. F. Molecular of on American Scholar), no insecticides have been directed against these channels. The limited phyletic specificity of the have promoted the that insect not be selectively this is with the that insect and vertebrate are with different to a of peptide toxins and chemical H. J. 1997; PubMed Scopus Google Scholar). of families of (4Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google Scholar, 10King G.F. Tedford H.W. Maggio F. J. Toxicol. Toxin Rev. 2002; 21: 359-389Crossref Google Scholar) that specific of insect is This is in the study, we demonstrated that ω-ACTX-Hv1a is lethal to a of insect but against heterologously expressed rat HVA concentrations as high as 10 The of specificity of ω-ACTX-Hv1a for insect it lead for the development of insecticides directed against insect calcium channels. In this study, we the of ω-ACTX-Hv1a to a template for rational design of such The cockroach neuronal binding assays and the of insect all identified three critical residues Asn27, and that we to the epitope of the toxin. is remarkable that these residues, in the form a spatially contiguous epitope when the three-dimensional structure of ω-ACTX-Hv1a Arg35 is the of this binding by and is one of the most residues J. 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King G.F. 2004; Scholar) has that of Australian funnel-web a of ω-ACTX-Hv1a in and in the of these the residues Pro10, Asn27, and are in all that have been to in H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (118) Google for of Thus, the available to that the of has been conserved in this family of of the ω-ACTX-Hv1a with on far as we are the toxin that have been in are on the cone toxins J.I. M. A. K. J. Biol. 1994; PubMed Google Scholar, M.J. R. J. Biol. 1997; PubMed Scopus Google Scholar, M.J. R. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar) and J.I. M. A. A. K. Biochem. PubMed Scopus Google Scholar, K. P. Ramachandran J. PubMed Scopus Google Scholar). of these which selectively block HVA currents by vertebrate a we in this for ω-ACTX-Hv1a R. J. PubMed Scopus Google for a the three residues that form the core of ω-ACTX-Hv1a Pro10, Asn27, and are spatially contiguous the three residues in the of and are and their are from one using the by in with ω-ACTX-Hv1a, of both and residues that to channel binding R. J. PubMed Scopus Google Scholar). The ω-ACTX-Hv1a be on a of the residues in of the consistent with that concentrations of ω-ACTX-Hv1a not block HVA currents by heterologously expressed rat channels. and are the most critical residues for block of vertebrate channels by the but these are not in the ω-ACTX-Hv1a We that the and ω-ACTX-Hv1a different of with their calcium channel ω-ACTX-Hv1a as a for of the calcium channel target of ω-ACTX-Hv1a in be to high of chemical to lead chemical insecticides on the of of Although has a in the it the of are limited in Eur. J. 2002; PubMed Scopus Google Scholar). chemical have a critical of the lead in insecticide and of all in the were or from J. Nat. 2003; PubMed Scopus Google Scholar). of the most insecticidal have been from including the and Sci. Scopus Google Scholar). Thus, a to high on of the target of ω-ACTX-Hv1a is rational design of insecticides on the In have been of rational design of on of a we to as Biochem. 2003; PubMed Scopus Google Scholar, Nat. Rev. 2004; PubMed Scopus Google Scholar). that the but spatially contiguous binding epitope on J.C. M.E. P. Science. 2002; PubMed Scopus Google Scholar). to the are three in which were to the of peptide toxins that block vertebrate calcium or channels J. PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). In the most study, the of was by with chemical to three functional residues and identified in alanine as the of is and residues such as are to be in the the had of μm against human S. PubMed Scopus Google Scholar). Although these channels with J.I. M. A. A. K. Biochem. PubMed Scopus Google Scholar, K. P. Ramachandran J. PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar), the have sufficient to for as S. PubMed Scopus Google Scholar). far as we are is no for synthetic of the of insecticidal peptide toxin with the of The for this is presumably of design ω-ACTX-Hv1a, the have been for three from the Australian funnel-web F. King G.F. J. Biol. 2002; PubMed Scopus Google Scholar) and insecticidal neurotoxins from the E. S. M. J. Biol. 1997; PubMed Scopus Google Scholar) and R. M. J. Biol. 2004; PubMed Scopus Google Scholar). ω-ACTX-Hv1a most a best for rational insecticide design this peptide is the of the and is The core ω-ACTX-Hv1a residues Pro10, Asn27, and to a contiguous of which the of a Biochem. 2003; PubMed Scopus Google Scholar). In the of a of R. M. J. Biol. 2004; PubMed Scopus Google Scholar), which a template for design of is that the of ω-ACTX-Hv1a development of a new rational design in insecticide a that to be is a the remarkable and phyletic specificity of the peptide toxin. not to be to insect functional a to binding to the vertebrate of the insect to this lead to with We for to were by the of which is in by the

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