ProTx-II(ProTx-2,ProtoxinII) isatoxinthatwasoriginallyisolatedfromThrixopelmapruriens(Peruviangreenvelvettarantula).ProTx-II inhibitsbothtetrodotoxin-sensitiveand tetrodotoxin-resistant voltage-gatedsodiumchannels.ProTx-II inhibitsactivationbyshiftingthevoltage-dependenceofchannelactivationtomorepositivepotentials. ProTx-IIblocksNav1.7 withanIC50 valueofaround300pM,Nav1.2, Nav1.5and Nav1.6 withIC50 valuesof41nM,79nMand26nMrespectively.ItalsoactsonCav3.1/CACNA1GandinteractsmoreweaklywiththerelatedT-TypechannelCav3.2/CACNA1HbutpotentlyinhibitstheL-typecalciumchannelCav1.2/CACNA1C.ProTx-II blocksactionpotentialpropagationinnociceptors.
ProTx-II#07PTX002efficientlyblocksthehumanNav1.7channel.Familiesoftransiently-expressedhumanNav1.7currenttracesincontrolandinthepresenceof1nMProTx-IIshowingmorethan50%currentinhibition.Currentswereevokedbydepolarizingpulsesfromaholdingpotentialof-80mVtosteppotentialvaryingfrom-60to30mV.TheIC50valueforcurrentinhibitionbyProTx-IIis0.31nM.
Fullbioassayresults
Description:
AAsequence: Tyr-Cys2-Gln-Lys-Trp-Met-Trp-Thr-Cys9-Asp-Ser-Glu-Arg-Lys-Cys15-Cys16-Glu-Gly-Met-Val-Cys21-Arg-Leu-Trp-Cys25-Lys-Lys-Lys-Leu-Trp-OH
Disulfidebonds: Cys2-Cys16,Cys9-Cys21,Cys15-Cys25
Length(aa): 30
Formula: C168H250N46O41S8
MolecularWeight: 3826.65Da
Appearance:Whitelyophilizedsolid
Solubility: waterorsalinebuffer
CASnumber: 484598-36-9
Source: Synthetic
Purityrate: >95%
Reference:
Spider-venompeptidesthattargetvoltage-gatedsodiumchannels:pharmacologicaltoolsandpotentialtherapeuticleads
Voltage-gatedsodium(Na(V))channelsplayacentralroleinthepropagationofactionpotentialsinexcitablecellsinbothhumansandinsects.ManyvenomousanimalshavethereforeevolvedtoxinsthatmodulatetheactivityofNa(V)channelsinordertosuBDuetheirpreyanddeterpredators.SpidervenomsinparticulararerichinNa(V)channelmodulators,withone-thirdofallknownionchanneltoxinsfromspidervenomsactingonNa(V)channels.Herewereviewthelandscapeofspider-venompeptidesthathavesofarbeendescribedtotargetvertebrateorinvertebrateNa(V)channels.Thesepeptidesfallinto12distinctfamiliesbasedontheirprimarystructureandcysteinescaffold.Someofthesepeptideshavebecomeusefulpharmacologicaltools,whileothershavepotentialastherapeuticleadsbecausetheytargetspecificNa(V)channelsubtypesthatareconsideredtobeimportantanalgesictargets.Spidervenomsareconservativelypredictedtocontainmorethan10millionbioactivepeptidesandsofaronly0.01%ofthisdiversitybeencharacterised.Thus,itislikelythatfutureresearchwillrevealadditionalstructuralclassesofspider-venompeptidesthattargetNa(V)channels.
KlintJK., etal. (2012)Spider-venompeptidesthattargetvoltage-gatedsodiumchannels:pharmacologicaltoolsandpotentialtherapeuticleads. Toxicon. PMID:22543187
EvidenceformultipleeffectsofProTxIIonactivationgatinginNa(V)1.5
ThepeptidetoxinProTxII,recentlyisolatedfromthevenomofthetarantulaspiderThrixopelmapruriens,modifiesgatinginvoltage-gatedNa+andCa2+channels.ProTxIIisdistinctfromotherknownNa+channelgatingmodifiertoxinsinthatitaffectsactivation,butnotinactivation.Itshiftsactivationgatingpositivelyanddecreasescurrentmagnitudesuchthatthedose-dependenceoftoxinactionmeasuredatasinglepotentialreflectsbotheffects.Totesttheextenttowhichtheseeffectswereindependent,wetrackedseveraldifferentmeasuresofcurrentamplitude,voltage-dependentactivation,andcurrentkineticsinNa(V)1.5inarangeoftoxinconcentrations.ChangesinvoltagedependenceandadecreaseinG(max)appearedatrelativelylowconcentrations(40-100nM)whileapositiveshiftinthevoltagerangeofactivationwasapparentathighertoxinconcentrations(>or=500nM).BecauseProTxIIcarriesanet+4chargewetestedwhetherelectrostaticinteractionscontributedtotoxinaction.WeexaminedtheeffectsofProTxIIinthepresenceofhighextracellularBa2+,knowntoscreenand/orbindtosurfacecharge.Some,butnotallaspectsofProTxIImodificationweresensitivetothepresenceofBa2+indicatingthecontributionofanelectrostatic,surfacecharge-likemechanismandsupportingtheideaofamulti-facetedtoxin-channelinteraction.
EdgertonG.B., etal. (2008)EvidenceformultipleeffectsofProTxIIonactivationgatinginNa(V)1.5, Toxicon. PMID:18657562
ProTx-II,aselectiveinhibitorofNav1.7sodiumchannels,blocksactionpotentialpropagationinnociceptors
Voltage-gatedsodium(Na(V)1)channelsplayacriticalroleinmodulatingtheexcitABIlityofsensoryneurons,andhumangeneticevidencepointstoNa(V)1.7asanessentialcontributortopainsignaling.Humanloss-of-functionmutationsinSCN9A,thegeneencodingNa(V)1.7,causechannelopathy-associatedindifferencetopain(CIP),whereasgain-of-functionmutationsareassociatedwithtwoinheritedpainfulneuropathies.AlthoughthehumangeneticdatamakeNa(V)1.7anattractivetargetforthedevelopmentofanalgesics,pharmacologicalproof-of-conceptinexperimentalpainmodelsrequiresNa(V)1.7-selectivechannelblockers.Here,weshowthatthetarantulavenompeptideProTx-IIselectivelyinteractswithNa(V)1.7channels,inhibitingNa(V)1.7withanIC(50)valueof0.3nM,comparedwithIC(50)valuesof30to150nMforotherheterologouslyexpressedNa(V)1subtypes.ThissubtypeselectivitywasabolishedbyapointmutationinDIIS3.ItisinterestingthatapplicationofProTx-IItodesheathedcutaneousnervescompletelyblockedtheC-fibercompoundactionpotentialatconcentrationsthathadlittleeffectonAbeta-fiberconduction.ProTx-IIapplicationhadlittleeffectonactionpotentialpropagationoftheintactnerve,whichmayexplainwhyProTx-IIwasnotefficaciousinrodentmodelsofacuteandinflammatorypain.Mono-iodo-ProTx-II((125)I-ProTx-II)bindswithhighaffinity(K(d)=0.3nM)torecombinanthNa(V)1.7channels.Bindingof(125)I-ProTx-IIisinsensitivetothepresenceofotherwellcharacterizedNa(V)1channelmodulators,suggestingthatProTx-IIbindstoanovelsite,whichmaybemoreconducivetoconferringsubtypeselectivitythanthesiteoccupiedbytrADItionallocalanestheticsandanticonvulsants.Thus,the(125)I-ProTx-IIbindingassay,describedhere,offersanewtoolinthesearchfornovelNa(V)1.7-selectiveblockers.
WilliamA., etal.(2007)ProTx-II,aselectiveinhibitorofNav1.7sodiumchannels,blocksactionpotentialpropagationinnociceptors. Mol.Pharm. PMID:18728100
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
Differentialphospholipidbindingbysite3andsite4toxins.Implicationsforstructuralvariabilitybetweenvoltage-sensitivesodiumchanneldomains
Ithasbeenshownrecentlythatpolypeptidetoxinsthatmodulatethegatingpropertiesofvoltage-sensitivecationchannelsareabletobindtophospholipidmembranes,leadingtothesuggestionthatthesetoxinsareabletoaccessachannel-bindingsitethatremainsmembrane-restricted(Lee,S.-Y.,andMacKinnon,R.(2004)Nature430,232-235).WethereforeexaminedtheabilityofanthopleurinB(ApB),aseaanemonetoxinthatselectivelymodifiesinactivationkineticsofNa(V)1.xchannels,andProTx-II,aspidertoxinthatmodifiesactivationkineticsofthesamechannels,tobindtoliposomes.WhereasProTx-IIcanbequantitativelydepletedfromsolutionuponincubationwithphosphatidylcholine/phosphatidylserineliposomes,ApBdisplaysnodiscernIBLephospholipidbindingactivity.Wethereforeexaminedtheactivitiesofstructurallyunrelatedsite3andsite4toxinsderivedfromLeiurusandCentruroidesvenoms,respectively,inthesameassay.LikeApB,thesite3toxinLqqVshowsnolipidbindingactivity,whereasthesite4toxinCentruroidestoxinII,likeProTx-II,iscompletelybound.WeconcludethattoxinsthatmodifyinactivationkineticsviabindingtoNa(V)1.xsite3lacktheabilitytobindphospholipids,whereassite4toxins,whichmodifyactivation,havethisactivity.ThisinherentdifferencesuggeststhattheconformationofdomainIImorecloselyresemblesthatoftheK(V)APchannelthandoestheconformationofdomainIV.
SmithJ.J., etal. (2005)Differentialphospholipidbindingbysite3andsite4toxins.Implicationsforstructuralvariabilitybetweenvoltage-sensitivesodiumchanneldomains, JBiolChem. PMID:15632158
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.
MiddletonR.E., etal. (2002)Twotarantulapeptidesinhibitactivationofmultiplesodiumchannels, Biochemistry. PMID:12475222
