HuwentoxinIV (HwTx-IV) isaneurotoxinthatwasoriginallyisolatedfromHaplopelmaschmidti(Chinesebirdspider).Thislethalneurotoxinactsselectivelyon tetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannels,withanIC50 of30nMinratDRGneurons.Itpreferentiallyinhibitsneuronal voltage-gatedsodiumchannelsubtypehNav1.7 (SCN9A,IC50 is26nM), rNav1.2 (SCN2A,IC50 is150nM),and rNav1.3 (SCN3A,IC50 is338nM),comparedwithmusclesubtypesrNav1.4(SCN4A)andhNav1.5(SCN5A)(IC50 is>10µM).HuwentoxinIV inhibitstheactivationofsodiumchannelsbytrappingthevoltagesensorofdomainIIofthesite4intheinward,closedconfiguration.Cy5-HuwentoxinIVisafluorescentlytaggedversionofHuwentoxinIV.Description:Productcode:N/A.Category:Sodiumchannels.Tags:nav,tetrodotoxin.AAsequence: Glu-Cys2-Leu-Glu-Ile-Phe-Lys-Ala-Cys9-Asn-Pro-Ser-Asn-Asp-Gln-Cys16-Cys17-Lys-Ser-Ser-Lys-Leu-Val-Cys24-Ser-Arg-Lys-Thr-Arg-Trp-Cys31-Lys-Tyr-Gln-Ile-NH2Disulfidebonds: Cys2-Cys17,Cys9-Cys24 andCys16-Cys31Length(aa): 35FormulaofHwTx-IV: C174H277N51O52S6MolecularWeightofHwTx-IV: 4107.20DaAppearance:darklyophilizedsolidSolubility: waterandsalinebufferCASnumber:Source: SyntheticPurityrate: >95%Cy5:λex646nm,λem662nmStoichiometry:1:1Reference:Analysisofthestructuralandmolecularbasisofvoltage-sensitivesodiumchannelinhibitionbythespidertoxin,Huwentoxin-IV(μ-TRTX-Hh2a)Voltage-gatedsodiumchannels(VGSCs)areessentialtothenormalfunctionofthevertebratenervoussystem.AberrantfunctionofVGSCsunderliesavarietyofdisorders,includingepilepsy,arrhythmia,andpain.Alargenumberofanimaltoxinstargettheseionchannelsandmayhavesignificanttherapeuticpotential.Mostofthesetoxins,however,havenotbeencharacterizedindetail.Here,bycombiningpatchclampelectrophysiologyandrADIoligandbindingstudieswithpeptidemutagenesis,NMRstructuredetermination,andmolecularmodeling,wehaverevealedkeymoleculardeterminantsoftheinteractionbetweenthetarantulatoxinhuwentoxin-IVandtwoVGSCisoforms,Nav1.7andNav1.2.Ninehuwentoxin-IVresidues(F6A,P11A,D14A,L22A,S25A,W30A,K32A,Y33A,andI35A)wereimportantforblockofNav1.7andNav1.2.Importantly,moleculardynamicssimulationsandNMRstudiesindicatedthatfoldingwasnormalforseveralkeymutants,suggestingthattheseaminoacidsprobablymakespecificinteractionswithsodiumchannelresidues.Additionally,weidentifiedseveralaminoacids(F6A,K18A,R26A,andK27A)thatareinvolvedinisoform-specificVGSCinteractions.Ourstructuralandfunctionaldatawereusedtomodelthedockingofhuwentoxin-IVintothedomainIIvoltagesensorofNav1.7.ThemodelpredictsthatahydrophobicpatchcomposedofTrp-30andPhe-6,alongwiththebasicLys-32residue,docksintoagrooveformedbytheNav1.7S1-S2andS3-S4loops.Theseresultsprovidenewinsightintothestructuralandmolecularbasisofsodiumchannelblockbyhuwentoxin-IVandmayprovideabasisfortherationaldesignoftoxin-basedpeptideswithimprovedVGSCpotencyand/orselectivity.MinassianNA.,etal.(2013)Analysisofthestructuralandmolecularbasisofvoltage-sensitivesodiumchannelinhibitionbythespidertoxin,Huwentoxin-IV(μ-TRTX-Hh2a). JBC. PMID: 23760503Commonmoleculardeterminantsoftarantulahuwentoxin-IVinhibitionofNa+channelvoltagesensorsindomainsIIandIVThevoltagesensorsofdomainsIIandIVofsodiumchannelsareimportantdeterminantsofactivationandinactivation,respectively.AnimaltoxinsthatalterelectrophysiologicalexcitABIlityofmusclesandneuronsoftenmodifysodiumchannelactivationbyselectivelyinteractingwithdomainIIandinactivationbyselectivelyinteractingwithdomainIV.Thissuggeststhattheremaybesubstantialdifferencesbetweenthetoxin-bindingsitesinthesetwoimportantdomains.Hereweexploretheabilityofthetarantulahuwentoxin-IV(HWTX-IV)toinhibittheactivityofthedomainIIandIVvoltagesensors.HWTX-IVisspecificfordomainII,andweidentifyfiveresiduesintheS1-S2(Glu-753)andS3-S4(Glu-811,Leu-814,Asp-816,andGlu-818)regionsofdomainIIthatarecrucialforinhibitionofactivationbyHWTX-IV.ThesedataindicatethatasingleresidueintheS3-S4linker(Glu-818inhNav1.7)iscrucialforallowingHWTX-IVtointeractwiththeotherkeyresiduesandtrapthevoltagesensorintheclosedconfiguration.MutagenesisanalysisindicatesthatthefivecorrespondingresiduesindomainIVareallcriticalforendowingHWTX-IVwiththeabilitytoinhibitfastinactivation.Ourdatasuggestthatthetoxin-bindingmotifindomainIIisconservedindomainIV.Increasingourunderstandingofthemoleculardeterminantsoftoxininteractionswithvoltage-gatedsodiumchannelsmaypermitdevelopmentofenhancedisoform-specificvoltage-gatingmodifiers.Xiao,Y., etal.(2011)Commonmoleculardeterminantsoftarantulahuwentoxin-IVinhibitionofNa+channelvoltagesensorsindomainsIIandIV, JBC. PMID: 21659528 ThetarantulatoxinsProTx-IIandhuwentoxin-IVdifferentiallyinteractwithhumanNav1.7voltagesensorstoinhibitchannelactivationandinactivationThevoltage-gatedsodiumchannelNa(v)1.7playsacrucialroleinpain,anddrugsthatinhibithNa(v)1.7mayhavetremendoustherapeuticpotential.ProTx-IIandhuwentoxin-IV(HWTX-IV),cystineknotpeptidesfromtarantulavenoms,preferentiallyblockhNa(v)1.7.Understandingtheinteractionsofthesetoxinswithsodiumchannelscouldaidthedevelopmentofnovelpaintherapeutics.WhereasbothProTx-IIandHWTX-IVhavebeenproposedtopreferentiallyblockhNa(v)1.7activationbytrappingthedomainIIvoltage-sensorintherestingconfiguration,weshowthatspecificresiduesinthevoltage-sensorpaddleofdomainIIplaysubstantiallydifferentrolesindeterminingtheaffinitiesofthesetoxinstohNa(v)1.7.ThemutationE818CincreasesProTx-II‘sandHWTX-IV‘sIC(50)forblockofhNa(v)1.7currentsby4-and400-fold,respectively.Incontrast,themutationF813GdecreasesProTx-IIaffinityby9-foldbuthasnoeffectonHWTX-IVaffinity.ItisnoteworthythatwealsoshowthatProTx-II,butnotHWTX-IV,preferentiallyinteractswithhNa(v)1.7toimpedefastinactivationbytrappingthedomainIVvoltage-sensorintherestingconfiguration.MutationsE1589QandT1590KindomainIVeachdecreasedProTx-II’sIC(50)forimpairmentoffastinactivationby~6-fold.IncontrastmutationsD1586AandF1592Aindomain-IVincreasedProTx-II’sIC(50)forimpairmentoffastinactivationby~4-fold.OurresultsshowthatwhereasProTx-IIandHWTX-IVbindingdeterminantsondomain-IImayoverlap,domainIIplaysamuchmorecrucialroleforHWTX-IV,andcontrarytowhathasbeenproposedtobeaguidingprincipleofsodiumchannelpharmacology,moleculesdonothavetoexclusivelytargetthedomainIVvoltage-sensortoinfluencesodiumchannelinactivation.Xiao,Y., etal.(2010)ThetarantulatoxinsProTx-IIandhuwentoxin-IVdifferentiallyinteractwithhumanNav1.7voltagesensorstoinhibitchannelactivationandinactivation, MolPharmacol. PMID: 20855463Mechanismofactionoftwoinsecttoxinshuwentoxin-IIIandhainantoxin-VIonvoltage-gatedsodiumchannelsSelenocosmiahuwenaandSelenocosmiahainanaaretwotarantulaspeciesfoundinsouthernChina.Theirvenomscontainabundantpeptidetoxins.Twonewneurotoxicpeptides,huwentoxin-III(HWTX-III)andhainantoxin-VI(HNTX-VI),wereobtainedfromthevenomusingion-exchangechromatographyandreverse-phasehighperformanceliquidchromatography(RP-HPLC).ThemechanismofactionofHWTX-IIIandHNTX-VIoninsectneuronalvoltage-gatedsodiumchannels(VGSCs)wasstudiedviawhole-cellpatchclamptechniques.Inafashionsimilartodelta-atracotoxins,HNTX-VIcaninduceaslowdownofcurrentinactivationoftheVGSCandreductioninthepeakofNa+currentincockroachdorsalunpairedmedian(DUM)neurons.Meanwhile,10micromol/LHNTX-IVcausedapositiveshiftofsteady-stateinactivationofsodiumchannel.HWTX-IIIinhibitedVGSCsonDUMneurons(concentrationoftoxinathalf-maximalinhibition(IC(50))approximately1.106micromol/L)inawaymuchsimilartotetrodotoxin(TTX).HWTX-IIIhadnoeffectonthekineticsofactivationandinactivation.Theshiftinthesteady-stateinactivationcurvewasdistinctfromotherdepressantspidertoxins.ThediverseeffectandthemechanismofactionofthetwoinsecttoxinsillustratethediverseBIOLOGicalactivitiesofspidertoxinsandprovideafreshtheoreticalfoundationtodesignanddevelopnovelinsecticides.WangRL, etal.(2010)Mechanismofactionoftwoinsecttoxinshuwentoxin-IIIandhainantoxin-VIonvoltage-gatedsodiumchannels. PMID: 20506577Tarantulahuwentoxin-IVinhibitsneuronalsodiumchannelsbybindingtoreceptorsite4andtrappingthedomainiivoltagesensorintheclosedconfigurationPeptidetoxinswithhighaffinity,divergentpharmacologicalfunctions,andisoform-specificselectivityarepowerfultoolsforinvestigatingthestructure-functionrelationshipsofvoltage-gatedsodiumchannels(VGSCs).Althoughanumberofinterestinginhibitorshavebeenreportedfromtarantulavenoms,littleisknownaboutthemechanismfortheirinteractionwithVGSCs.Weshowthathuwentoxin-IV(HWTX-IV),a35-residuepeptidefromtarantulaOrnithoctonushuwenavenom,preferentiallyinhibitsneuronalVGSCsubtypesrNav1.2,rNav1.3,andhNav1.7comparedwithmusclesubtypesrNav1.4andhNav1.5.OfthefiveVGSCsexamined,hNav1.7wasmostsensitivetoHWTX-IV(IC(50)approximately26nM).Followingapplicationof1micromHWTX-IV,hNav1.7currentscouldonlybeelicitedwithextremedepolarizations(>+100mV).RecoveryofhNav1.7channelsfromHWTX-IVinhibitioncouldbeinducedbyextremedepolarizationsormoderatedepolarizationslastingseveralminutes.Site-directedmutagenesisanalysisindicatedthatthetoxindockedatneurotoxinreceptorsite4locatedattheextracellularS3-S4linkerofdomainII.MutationsE818QandD816NinhNav1.7decreasedtoxinaffinityforhNav1.7byapproximately300-fold,whereasthereversemutationsinrNav1.4(N655D/Q657E)andthecorrespondingmutationsinhNav1.5(R812D/S814E)greatlyincreasedthesensitivityofthemuscleVGSCstoHWTX-IV.Ourdataidentifyanovelmechanismforsodiumchannelinhibitionbytarantulatoxinsinvolvingbindingtoneurotoxinreceptorsite4.Incontrasttoscorpionbeta-toxinsthattraptheIIS4voltagesensorinanoutwardconfiguration,weproposethatHWTX-IVtrapsthevoltagesensorofdomainIIintheinward,closedconfiguration.Xiao,Y., etal.(2008)Tarantulahuwentoxin-IVinhibitsneuronalsodiumchannelsbybindingtoreceptorsite4andtrappingthedomainiivoltagesensorintheclosedconfiguration, JBC. PMID: 18628201Functionandsolutionstructureofhuwentoxin-IV,apotentneuronaltetrodotoxin(TTX)-sensitivesodiumchannelantagoNISTfromChinesebirdspiderSelenocosmiahuwenaWehaveisolatedahighlypotentneurotoxinfromthevenomoftheChinesebirdspider,Selenocosmiahuwena.This4.1-kDatoxin,whichhasbeennamedhuwentoxin-IV,contains35residueswiththreedisulfidebridges:Cys-2-Cys-17,Cys-9-Cys-24,andCys-16-Cys-31,assignedbyachemicalstrategyincludingpartialreductionofthetoxinandsequenceanalysisofthemodifiedintermediates.Itspecificallyinhibitstheneuronaltetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannelwiththeIC(50)valueof30nminadultratdorsalrootganglionneurons,whilehavingnosignificanteffectonthetetrodotoxin-resistant(TTX-R)voltage-gatedsodiumchannel.ThistoxinseemstobeasiteItoxinaffectingthesodiumchannelthroughamechanismquitesimilartothatofTTX:itsuppressesthepeaksodiumcurrentwithoutalteringtheactivationorinactivationkinetics.Thethree-dimensionalstructureofhuwentoxin-IVhasbeendeterminedbytwo-dimensional(1)HNMRcombinedwithdistantgeometryandsimulatedannealingcalculationbyusing527nuclearOverhausereffectconstraintsand14dihedralconstraints.Theresultingstructureiscomposedofadouble-strandedantiparallelbeta-sheet(Leu-22-Ser-25andTrp-30-Tyr-33)andfourturns(Glu-4-Lys-7,Pro-11-Asp-14,Lys-18-Lys-21andArg-26-Arg-29)andbelongstotheinhibitorcystineknotstructuralfamily.Aftercomparisonwithothertoxinspurifiedfromthesamespecies,weareconvincedthatthepositivelychargedresiduesofloopIV(residues25-29),especiallyresidueArg-26,mustbecrucialtoitsbindingtotheneuronaltetrodotoxin-sensitivevoltage-gatedsodiumchannel.Peng,K., etal.(2002)Functionandsolutionstructureofhuwentoxin-IV,apotentneuronaltetrodotoxin(TTX)-sensitivesodiumchannelantagonistfromChinesebirdspiderSelenocosmiahuwena, JBiolChem.PMID: 12228241Smartox"scitation:AnaturalpointmutationchangesbothtargetselectivityandmechanismofactionofseaanemonetoxinsAPETx3,anovelpeptideisolatedfromtheseaanemoneAnthopleuraelegantissima,isanaturallyoccurringmutantfromAPETx1,onlydifferingbyaThrtoProsubstitutionatposition3.APETx1isbelievedtobeaselectivemodulatorofhumanether-á-go-gorelatedgene(hERG)potassiumchannelswithaK(d)of34nM.Inthisstudy,APETx1,2,and3havebeensubjectedtoanelectrophysiologicalscreeningonawiderangeof24ionchannelsexpressedinXenopuslaevisoocytes:10clonedvoltage-gatedsodiumchannels(Na(V)1.2-Na(V)1.8,theinsectchannelsDmNa(V)1,BgNa(V)1-1a,andthearachnidchannelVdNa(V)1)and14clonedvoltage-gatedpotassiumchannels(K(V)1.1-K(V)1.6,K(V)2.1,K(V)3.1,K(V)4.2,K(V)4.3,K(V)7.2,K(V)7.4,hERG,andtheinsectchannelShakerIR).Surprisingly,theThr3ProsubstitutionresultsinacompleteabolishmentofAPETx3modulationonhERGchannelsandprovidesthistoxintheabilitytobecomeapotent(EC(50)276nM)modulatorofvoltage-gatedsodiumchannels(Na(V)s)becauseitslowsdowntheinactivationofmammalianandinsectNa(V)channels.OurstudyalsoshowsthatthehomologoustoxinsAPETx1andAPETx2displaypromiscuouspropertiessincetheyarealsocapableofrecognizingNa(V)channelswithIC(50)valuesof31nMand114nM,respectively,causinganinhibitionofthesodiumconductancewithoutaffectingtheinactivation.Ourresultsprovidenewinsightsinkeyresiduesthatallowtheseseaanemonetoxinstorecognizedistinctionchannelswithsimilarpotencybutwithdifferentmodulatoryeffects.FurThermore,wedescribeforthefirsttimethetargetpromiscuityofafamilyofseaanemonetoxinsthusfarbelievedtobehighlyselective.PeigneurS, etal. (2012)Anaturalpointmutationchangesbothtargetselectivityandmechanismofactionofseaanemonetoxins. FASEBJ. PMID: 22972919