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. ProTx-I isavaluabletooltodiscriminatebetweenCav3.1andCav3.2A,B,C,RecordingtracesofhNav1.7currentinthepresenceofSmartoxsProTx-I(100and300nM).Thecurrentwaselicitedbya50ms-depolarizingpulseto-10mVfromaholdingpotentialof-90mV.Inter-sweepperiodwas10s.Currentamplitudeswereplottedagainsttime.Blackbarindicatestoxinapplication.D,FamiliesofhNav1.7currenttracesincontrolandinthepresenceof100nMProTx-I.Currentswereevokedbydepolarizingpulsesfrom-60mVto40mV,whilethecellwasholdat-90mV.E,Amplitude-voltagerelationshipsobtainedfromD.Description:Productcode:N/A.Categories:Calciumchannels,Lowvoltage-gatedCa2+channels,Sodiumchannels.Tags:Cav3.1,Nav1.7,nav1.8,protox,protoxin,t-type,tetrodotoxin,TRPA1,ttx.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-OHDisulfidebridges:Cys2-Cys16,Cys9-Cys21,Cys15-Cys28Length(aa):35Formula:C171H245N53O47S6MolecularWeight:3987.50DaAppearance:WhitelyophilizedsolidSolubility:waterorsalinebufferCASnumber:NotavailableSource:SyntheticPurityrate:>95%Reference:TwotarantulapeptidesinhibitactivationofmultiplesodiumchannelsTwopeptides,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: 12475222ProTx-IandProTx-II:gatingmodifiersofvoltage-gatedsodiumchannelsThetarantulavenompeptidesProTx-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: 17087985TarantulatoxinProTx-IdifferentiatesbetweenhumanT-typevoltage-gatedCa2+ChannelsCav3.1andCav3.2ProTx-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: 20351484ATarantula-VenomPeptideAntagonizestheTRPA1NociceptorIonChannelbyBindingtotheS1-S4GatingDomainBACKGROUND: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