ProtoxinI(ProTx-I;β-theraphotoxin-Tp1a) isatoxinthatwasoriginallyisolatedfromthevenomofThrixopelmapruriens(Peruviangreenvelvettarantula).ThistoxinreversIBLyinhibitsthetetrodotoxin(TTX)-resistantchannel Nav1.8(IC50 =27nM)and Nav1.2,Nav1.5andNav1.7 withIC50 valuesbetween50and100nM.FurThermore, ProTx-I shiftsthevoltagedependenceactivityof T-typeCav3.1channels (IC50=50nM)withoutaffectingthevoltagedependenceofinactivation.Biotin-ProTx-Iisabiotin-taggedversionofProTx-I.
Description:
AAsequence:Glu-Cys2-Arg-Tyr-Trp-Leu-Gly-Gly-Cys9-Ser-Ala-Gly-Gln-Thr-Cys15-Cys16-Lys-His-Leu-Val-Cys21-Ser-Arg-Arg-His-Gly-Trp-Cys28-Val-Trp-Asp-Gly-Thr-Phe-Ser-OH
Disulfidebridges:Cys2-Cys16,Cys9-Cys21,Cys15-Cys28
Length(aa):35
FormulaofProTx-I:C171H245N53O47S6
MolecularWeightofProTx-I:3987.50Da
Tag:Biotin
Appearance:Whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:Notavailable
Source:Synthetic
Purityrate:>95%
Reference:
Twotarantulapeptidesinhibitactivationofmultiplesodiumchannels
Twopeptides,ProTx-IandProTx-II,fromthevenomofthetarantulaThrixopelmapruriens,havebeenisolatedandcharacterized.ThesepeptideswerepurifiedonthebasisoftheirABIlitytoreversiblyinhibitthetetrodotoxin-resistantNachannel,Na(V)1.8,andareshowntobelongtotheinhibitorycystineknot(ICK)familyofpeptidetoxinsinteractingwithvoltage-gatedionchannels.Thefamilyhasseveralhallmarks:cystinebridgeconnectivity,mechanismofchannelinhibition,andpromiscuityacrosschannelswithinandacrosschannelfamilies.ThecystinebridgeconnectivityofProTx-IIisverysimilartothatofothermembersofthisfamily,i.e.,C(2)toC(16),C(9)toC(21),andC(15)toC(25).Thesepeptidesarethefirsthigh-affinityligandsfortetrodotoxin-resistantperipheralnerveNa(V)channels,butalsoinhibitotherNa(V)channels(IC(50)’s<100nM).ProTx-IandProTx-IIshiftthevoltagedependenceofactivationofNa(V)1.5tomorepositivevoltages,similartoothergating-modifierICKfamilymembers.ProTx-IalsoshiftsthevoltagedependenceofactivationofCa(V)3.1(alpha(1G),T-type,IC(50)=50nM)withoutaffectingthevoltagedependenceofinactivation.Toenablefurtherstructuralandfunctionalstudies,syntheticProTx-IIwasmade;itadoptsthesamestructureandhasthesamefunctionalpropertiesasthenativepeptide.SyntheticProTx-Iwasalsomadeandexhibitsthesamepotencyasthenativepeptide.SyntheticProTx-I,butnotProTx-II,alsoinhibitsK(V)2.1channelswith10-foldlesspotencythanitspotencyonNa(V)channels.ThesepeptidesrepresentnoveltoolsforexploringthegatingmechanismsofseveralNa(V)andCa(V)channels.
MiddeltonR.E, etal. (2002)Twotarantulapeptidesinhibitactivationofmultiplesodiumchannels.Biochemestry.PMID: 12475222
ProTx-IandProTx-II:gatingmodifiersofvoltage-gatedsodiumchannels
ThetarantulavenompeptidesProTx-IandProTx-IIinhibitvoltage-gatedsodiumchannelsbyshiftingtheirvoltagedependenceofactivationtoamorepositivepotential,thusactingbyamechanismsimilartothatofpotassiumchannelgatingmodifierssuchashanatoxinandVSTX1.ProTx-IandProTx-IIinhibitallsodiumchannel(Nav1)subtypestestedwithsimilarpotencyandrepresentthefirstpotentpeptidylinhibitorsofTTX-resistantsodiumchannels.Likegatingmodifiersofpotassiumchannels,ProTx-IandProTx-IIconformtotheinhibitorycystineknotmotif,andProTx-IIwasdemonstratedtobindtosodiumchannelsintheclosedstate.Bothtoxinshavebeensynthesizedchemically,andProTx-II,producedbyrecombinantmeans,hasbeenusedtomaptheinteractionsurfaceofthepeptidewiththeNav1.5channel.Incomparison,beta-scorpiontoxinsactivatesodiumchannelsbyshiftingthevoltagedependenceofactivationtomorenegativepotentials,andtogetherthesepeptidesrepresentvaluabletoolsforexploringthegatingmechanismofsodiumchannels.
PriestB.T., etal.(2007)ProTx-IandProTx-II:gatingmodifiersofvoltage-gatedsodiumchannels. Toxicon.PMID: 17087985
TarantulatoxinProTx-IdifferentiatesbetweenhumanT-typevoltage-gatedCa2+ChannelsCav3.1andCav3.2
ProTx-Ipeptide,avenomtoxinofthetarantulaThrixopelmapruriens,hasbeenreportedtointeractwithvoltage-gatedionchannels.ProTx-IreducedBa(2+)currentsthroughrecombinanthumanT-typevoltage-gatedCa(2+)channels,Ca(v)3.1(hCa(v)3.1),withroughly160-foldmorepotencythanthroughhCa(v)3.2channels.Chimericchannelproteins(hCa(v)3.1/S3S4andhCa(v)3.2/S3S4)wereproducedbyexchangingfourteenaminoacidsinthehCa(v)3.1domainIVS3-S4linkerregionandthecorrespondingregionofhCa(v)3.2betweeneachother.TheProTx-IsensitivitywasmarkedlyreducedinthehCa(v)3.1/S3S4chimeraascomparedtotheoriginalhCa(v)3.1channel,whilethehCa(v)3.2/S3S4chimeraexhibitedgreaterProTx-IsensitivitythantheoriginalhCa(v)3.2channel.TheseresultssuggestthatthedomainIVS3-S4linkerinthehCa(v)3.1channelmaycontainresiduesinvolvedintheinteractionofProTx-IwithT-typeCa(2+)channels.
OhkuboT, etal. (2010)TarantulatoxinProTx-IdifferentiatesbetweenhumanT-typevoltage-gatedCa2+ ChannelsCav3.1andCav3.2. JPharmacolSci. PMID: 20351484
ATarantula-VenomPeptideAntagonizestheTRPA1NociceptorIonChannelbyBindingtotheS1-S4GatingDomain
BACKGROUND:
Thevenomsofpredatorshavebeenanexcellentsourceofdiversehighlyspecificpeptidestargetingionchannels.HerewedescribethefirstknownpeptideantagoNISTofthenociceptorionchanneltransientreceptorpotentialankyrin1(TRPA1).
RESULTS:
WeconstructedarecombinantCDNAlibraryencoding∼100diverseGPI-anchoredpeptidetoxins(t-toxins)derivedfromspidervenomsandscreenedthislibrarybycoexpressioninXenopusoocyteswithTRPA1.Thisscreenresultedinidentificationofprotoxin-I(ProTx-I),a35-residuepeptidefromthevenomofthePeruviangreen-velvettarantula,Thrixopelmapruriens,asthefirstknownhigh-affinitypeptideTRPA1antagonist.ProTx-Iwaspreviouslyidentifiedasanantagonistofvoltage-gatedsodium(NaV)channels.Weconstructedat-toxinlibraryofProTx-Ialanine-scanningmutantsandscreenedthislibraryagainstNaV1.2andTRPA1.ThisrevealeddistinctpartiallyoverlappingsurfacesofProTx-Ibywhichitbindstothesetwoionchannels.Importantly,thismutagenesisyieldedtwonovelProTx-IvariantsthatareonlyactiveagainsteitherTRPA1orNaV1.2.Bytestingitsactivityagainstchimericchannels,weidentifiedtheextracellularloopsoftheTRPA1S1-S4gatingdomainastheProTx-Ibindingsite.
CONCLUSIONS:
Thesestudiesestablishourapproach,whichweterm“toxineering,”asagenerallyapplicablemethodforisolationofnovelionchannelmodifiersanddesignofionchannelmodifierswithalteredspecificity.TheyalsosuggestthatProTx-IwillbeavaluablepharmacologicalreagentforaddressingbiophysicalmechanismsofTRPA1gatingandthephysiologyofTRPA1functioninnociceptors,aswellasforpotentialclinicalapplicationinthecontextofpainandinflammation.
GuiJ,etal.(2014)ATarantula-VenomPeptideAntagonizestheTRPA1NociceptorIonChannelbyBindingtotheS1-S4GatingDomain. CurrBiol. PMID: 24530065
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