Hainantoxin-IV(HNTX-IV)isapeptidethatwasoriginallyisolatedfromthevenomoftheChinesebirdspiderOrnithoctonushainanaLiang(SelenocosmiahainanaLiang).IthasbeenreportedthatthispeptideisapotentantagoNISToftetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannels(VGSCs).Hainantoxin-IVbindstoTTX-SwithanIC50valueof34nMinadultratdorsalrootganglion(DRG)neurons.Tetrodotoxin-resistant(TTX-R)voltage-gatedsodiumchannelsarenotaffectedbyHainantoxin-IV.Itprobablyinteractswiththesite1throughamechanismquitesimilartothatofTTXwithoutaffectingtheactivationandinactivationkinetics.Description:Productcode:N/A.Category:Sodiumchannels.Tags:nav,tetrodotoxin,ttx.AAsequence:Glu-Cys2-Leu-Gly-Phe-Gly-Lys-Gly-Cys9-Asn-Pro-Ser-Asn-Asp-Gln-Cys16-Cys17-Lys-Ser-Ser-Asn-Leu-Val-Cys24-Ser-Arg-Lys-His-Arg-Trp-Cys31-Lys-Tyr-Glu-Ile-NH2(DisulfidebondsbetweenCys2-Cys17,Cys9-Cys24,andCys16-Cys31)Length(aa):35Formula:C166H257N53O50S6MolecularWeight:3987.6DaAppearance:WhitelyophilizedsolidSolubility:waterandsalinebufferCASnumber:NotavailableSource:SyntheticPurityrate:>97%Reference:Apositivelychargedsurfacepatchisimportantforhainantoxin-IVbindingtovoltage-gatedsodiumchannelsHainantoxin-IV(HNTX-IV),isolatedfromthevenomofthespiderOrnithoctonushainana,isaspecificantagonistoftetrodotoxin-sensitive(TTX-S)voltage-gatedsodiumchannelsinratdorsalrootganglion(DRG)cells.Itadoptsaninhibitorcystineknotmotif,andstructuralanalysisrevealedapositivelychargedpatchconsistingofArg26,Lys27,His28,Arg29andLys32distributedonitsmolecularsurface.OurpreviousstudydemonstratedthatLys27andArg29butnotArg26werecriticalresiduesforHNTX-IVbindingtoTTX-Ssodiumchannels.Inthepresentstudy,weexaminedtherolesofHis28andLys32intheinteractionofHNTX-IVwithitstarget.Twomutants,HNTX-IV-H28DandHNTX-IV-K32A,weregeneratedbysolid-phasechemicalsynthesisandpurifiedbyreverse-phaseHPLCafterrefoldingandoxidation,yieldingtwocompoundsofhighpuritywithmonoisotopicmassesof3962.66and3927.70 Da,respectively,asdeterminedbyMALDI-TOFmassspectrometry.Thisindicatedthepresenceofsixcysteineresiduesformingthreedisulfidebonds.Moreover,circulardichroismspectroscopyanalysisdemonstratedthatthesubstitutionofHis28orLys32didnotaffecttheoverallstructureofHNTX-IV.TheinhibitoryactivityofHNTX-IV-H28DandHNTX-IV-K32AagainstTTX-SsodiumchannelsinratDRGcellswasanalyzedbywhole-cellpatch-clamptechnique.TheIC(50)valuesforthemutantswere0.57and5.80 μM(17-foldand170-foldlowerthantheactivityofthenativetoxin),indicatingthatHis28andLys32maybeimportantfortheinhibitoryactivityofHNTX-IV.Takentogether,ourresultssuggestthatthepositivelychargedpatchmightbethebindingsitefortheinteractionofHNTX-IVwithTTX-Ssodiumchannels.ThesefindingsmightcontributetotheelucidationofthestructureandfunctionrelationshipofHNTX-IV.LiuY,etal.(2012)Apositivelychargedsurfacepatchisimportantforhainantoxin-IVbindingtovoltage-gatedsodiumchannels.JPeptSci.PMID:22927181Structure--activityrelationshipsofhainantoxin-IVandstructuredeterminationofactiveandinactivesodiumchannelblockers.Hainantoxin-IV(HNTX-IV)canspecificallyinhibittheneuronaltetrodotoxin-sensitivesodiumchannelsanddefinesanewclassofdepressantspidertoxin.ThesequenceofnativeHNTX-IVisECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH(2).Inthepresentstudy,toobtainfurtherinsightintotheprimaryandtertiarystructuralrequirementsofneuronalsodiumchannelblockers,wedeterminedthesolutionstructureofHNTX-IVasatypicalinhibitorcystineknotmotifandsynthesizedfourmutantsdesignedbasedonthepredictedsitesfollowedbystructuralelucidationoftwoinactivemutants.PharmacologicalstudiesindicatedthattheS12AandR26AmutantshadactivitiesnearthatofnativeHNTX-IV,whileK27AandR29Ademonstratedactivitiesreducedby2ordersofmagnitude.(1)HMRanalysisshowedthesimilarmolecularconformationsfornativeHNTX-IVandfoursyntheticmutants.FurThermore,inthedeterminedstructuresofK27AandR29A,thesidechainsofresidues27and29werelocatedintheidenticalspatialpositiontothoseofnativeHNTX-IV.TheseresultssuggestedthatresiduesSer(12),Arg(26),Lys(27),andArg(29)werenotresponsIBLeforstABIlizingthedistinctconformationofHNTX-IV,butLys(27)andArg(29)werecriticalforthebioactivities.ThepotencyreductionsproducedbyAlasubstitutionswereprimarilyduetothedirectinteractionoftheessentialresiduesLys(27)andArg(29)withsodiumchannelsratherthantoaconformationalchange.Aftercomparisonofthesestructuresandactivitieswithcorrelatedtoxins,wehypothesizedthatresiduesLys(27),Arg(29),His(28),Lys(32),Phe(5),andTrp(30)clusteredononefaceofHNTX-IVwereresponsibleforligandbinding.LiD,etal.(2004)Structure–activityrelationshipsofhainantoxin-IVandstructuredeterminationofactiveandinactivesodiumchannelblockers.JBiolChem.PMID:15201273Isolationandcharacterizationofhainantoxin-IV,anovelantagonistoftetrodotoxin-sensitivesodiumchannelsfromtheChinesebirdspiderSelenocosmiahainana.Aneurotoxin,namedhainantoxin-IV,waspurifiedfromthevenomofthespiderSelenocosmiahainana.TheaminoacidsequencewasdeterminedbyEdmandegradation,revealingittobea35-residuepolypeptideamidatedatitsCterminalandincludingthreedisulfidebridges:Cys2-Cys17,Cys9-Cys24,andCys16-Cys31assignedbypartialreductionandsequenceanalysis.Hainantoxin-IVshares80%sequenceidentitywithhuwentoxin-IVfromthespiderS.huwena,apotentantagonistthatactsatsite1ontetrodotoxin-sensitive(TTX-S)sodiumchannels,suggestingthathainantoxin-IVadoptsaninhibitorcystineknotstructuralmotiflikehuwentoin-IV.Underwhole-cellvoltage-clampconditions,thistoxinhasnoeffectontetrodotoxin-resistantvoltage-gatedsodiumchannelsinadultratdorsalrootganglionneurons,whileitblocksTTX-Ssodiumchannelsinamannersimilartohuwentoxin-IV,andtheactionsofbothtoxinsonsodiumcurrentsareverysimilartothatoftetrodotoxin.Thus,theydefineanewfamilyofspidertoxinsaffectingsodiumchannels.LiuZ,etal.(2003)Isolationandcharacterizationofhainantoxin-IV,anovelantagonistoftetrodotoxin-sensitivesodiumchannelsfromtheChinesebirdspiderSelenocosmiahainana.CellMolLifeSci.PMID:12827284Inhibitionofneuronaltetrodotoxin-sensitiveNa+channelsbytwospidertoxins:hainantoxin-IIIandhainantoxin-IV.Hainantoxin-IIIandhainantoxin-IV,isolatedfromthevenomoftheChinesebirdspiderSeleconosmiahainana,areneurotoxicpeptidescomposedof33-35residueswiththreedisulfidebonds.Usingwhole-cellpatch-clamptechnique,weinvestigatedtheiractiononionicchannelsofadultratdorsalrootganglionneurons.ItwasfoundthatthetwotoxinsdidnotaffectCa2+channels(bothhighvoltageactivatedandlowvoltageactivatedtypes)nortetrodotoxin-resistantvoltage-gatedNa+channels(VGSCs).However,hainantoxin-IIIandhainantoxin-IVstronglydepressedtheamplitudeoftetrodotoxin-sensitiveNa+currentswithIC50valuesof1.1and44.6nM,respectively.Bothhainantoxin-III(1nM)andhainantoxin-IV(50nM)causedahyperpolarizingshiftofabout10mVinthevoltagemidpointofsteady-stateNa+channelinactivation,buttheyshoweddifferenceinthereprimekineticsofVGSCs:hainantoxin-IIIsignificantlydecreasedtherecoveryratefrominactivationataprepulsepotentialof-80mVwhilehainantoxin-IVdidnotdo.Itisinterestingtonotethatsimilartohuwentoxin-IV,thetwohainantoxinsdidnotaffecttheactivationandinactivationkineticsofNa+currentsandataconcentrationof1microMtheycompletelyinhibitedtheslowinginactivationcurrentsinducedbyBMK-I(toxinIfromthescorpionButhusmartensiKarsch),ascorpionalpha-liketoxin.Theresultsindicatethathainantoxin-IIIandhainantoxin-IVarenovelspidertoxinsandaffectthemammalneuralNa+channelsthroughamechanismquitedifferentfromotherspidertoxinstargetingtheneuralreceptorsite3,suchasdelta-aractoxinsandmu-agatoxins.XiaoY,etal.(2003)Inhibitionofneuronaltetrodotoxin-sensitiveNa+channelsbytwospidertoxins:hainantoxin-IIIandhainantoxin-IV.EurJPharmacol.PMID:14512091Determinationofdisulfidebridgesoftwospidertoxins:Hainantoxin-IIIandHainantoxin-IVPeptidetoxinsareusuallyhighlybridgedproteinswithmultipairsofintrachaindisulfidebonds.Analysisofdisulfideconnectivityisanimportantfacetofproteinstructuredetermination.Inthispaper,wesuccessfullyassignedthedisulfidelinkageoftwonovelpeptidetoxins,calledHNTX-IIIandHNTX-IV,isolatedfromthevenomofOrnithoctonushainanaspider.BothpeptidesareusefulinhibitorsofTTX-sensitivevoltage-gatedsodiumchannelsandarecomposedofsixcysteineresiduesthatformthreedisulfidebonds,respectively.Firstly,thepeptideswerepartiallyreducedbytris(2-carboxyethyl)-phosphine(TCEP)in0.1Mcitratebuffercontaining6Mguanidine-HClat40°Cfortenminutes.Subsequently,thepartiallyreducedintermediatescontainingfreethiolswereseparatedbyreversed-phasehigh-performanceliquidchromatography(RP-HPLC)andalkylatedbyrapidcarboxamidomethylation.Then,thedisulfidebondsoftheintermediateswereanalyzedbyEdmandegradation.Byusingthestrategyabove,disulfidelinkagesofHNTX-IIIandHNTX-IVweredeterminedasI-IV,II-VandIII-VIpattern.Inaddition,thisstudyalsoshowedthatthismethodmayhaveagreatpotentialfordeterminingthedisulfidebondsofspiderpeptidetoxins.WangW.,etal.(2009)Determinationofdisulfidebridgesoftwospidertoxins:Hainantoxin-IIIandHainantoxin-IV.JVenomAnimToxinsinclTropDis.