BlockerofhighconductanceCa2+-activatedK+channel
Iberiotoxin(IbTx)isatoxinthatwasoriginallyisolatedfromButhustamulusscorpionvenom.IberiotoxininhibitsselectivelythehighconductanceCa2+-activatedK+channel(KCa1.1)atnanomolarconcentrations(IC50~2nM).Thistoxindoesnotaffectothertypesofcalcium-dependentorvoltage-dependentK+channels.IberiotoxinisavaluabletooltostudyspecificallyMaxi-Kchannels.
RecentlyquotedinBr.J.Pharmacol.
Description:
AAsequence:pGlu-Phe-Thr-Asp-Val-Asp-Cys-Ser-Val-Ser-Lys-GIu-Cys-Trp-Ser-Val-Cys-Lys-Asp-Leu-Phe-Gly-Val-Asp-Arg-Gly-Lys-Cys-Met-Gly-Lys-Lys-Cys-Arg-Cys-Tyr-GIn-OH
Disulfidebridges:Cys7-Cys28,Cys13-Cys33,Cys17-Cys35
Length(aa):37
Formula:C179H274N50O55S7
MolecularWeight:4230.8Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:129203-60-7
Source:Synthetic
Purityrate:>95%
Reference:
EffectsofthenovelBK(KCa1.1)channelopenerGoSlo-SR-5-130aredependentonthepresenceofBKβsubunits.
GoSlo-SRcompoundsareefficaciousBK(KCa1.1)channelopeners,butlittleisknownabouttheirmechanismofactionoreffectonbladdercontractility.WeexaminedtheeffectsoftwocloselyrelatedcompoundsonBKcurrentsandbladdercontractions.Acombinationofelectrophysiology,molecularBIOLOGyandsyntheticchemistrywasusedtoexaminetheeffectsoftwonovelchannelagoNISTsonBKchannelsfrombladdersmoothmusclecellsandinHEKcellsexpressingBKαaloneorincombinationwitheitherβ1orβ4subunits.GoSlo-SR-5-6shiftedthevoltagerequiredforhalfmaximalactivation(V1/2)ofBKchannelsapproximately-100 mV,irrespectiveofthepresenceofregulatoryβsubunits.Thedeaminatedderivative,GoSlo-SR-5-130,alsoshiftedtheactivationV1/2insmoothmusclecellsbyapproximately-100 mV;however,thiswasreducedby∼80%inHEKcellsexpressingonlyBKαsubunits.Whenβ1orβ4subunitswereco-expressedwithBKα,efficacywasrestored.GoSlo-SR-5-130causedaconcentration-dependentreductioninspontaneousbladdercontractionamplitudeandthiswasabolishedbyiberiotoxin,consistentwithaneffectonBKchannels.GoSlo-SR-5-130requiredβ1orβ4subunitstomediateitsfulleffects,whereasGoSlo-SR-5-6workedequallywellintheabsenceorpresenceofβsubunits.GoSlo-SR-5-130inhibitedspontaneousbladdercontractionsbyactivatingBKchannels.ThenovelBKchannelopener,GoSlo-SR-5-130,isapproximatelyfivefoldmoreefficaciousonBKchannelswithregulatoryβsubunitsandmaybeausefulscaffoldinthedevelopmentofdrugstotreatdiseasessuchasoveractivebladder.
LargeRJ.,etal.(2015)EffectsofthenovelBK(KCa1.1)channelopenerGoSlo-SR-5-130aredependentonthepresenceofBKβsubunits.BJP.PMID:25598230
Purificationandcharacterizationofaunique,potent,peptidylprobeforthehighconductancecalcium-activatedpotassiumchannelfromvenomofthescorpionButhustamulus
Aninhibitorofthehighconductance,Ca2(+)-activatedK+channel(PK,Ca)hasbeenpurifiedtohomogeneityfromvenomofthescorpionButhustamulusbyacombinationofionexchangeandreversed-phasechromatography.Thispeptide,whichhasbeennamediberiotoxin(IbTX),isoneoftwominorcomponentsofthecrudevenomwhichblocksPK,Ca.IbTXconsistsofasingle4.3-kDapolypeptidechain,asdeterminedbypolyacrylamidegelelectrophoresis,analysisofaminoacidcomposition,andEdmandegradation.Itscompleteaminoacidsequencehasbeendefined.IbTXdisplays68%sequencehomologywithcharyBDotoxin(ChTX),anotherscorpion-derivedpeptidylinhibitorofPK,Ca,and,likethislattertoxin,itsaminoterminuscontainsapyroglutamicacidresidue.However,IbTXpossesses4moreacidicand1lessbasicaminoacidresiduethandoesChTX,makingthistoxinmuchlesspositivelychargedthantheotherpeptide.Insinglechannelrecordings,IbTXreversIBLyblocksPK,Cainexcisedmembranepatchesfrombovineaorticsmoothmuscle.ItactsexclusivelyattheouterfaceofthechannelandfunctionswithanIC50ofabout250pM.BlockofchannelactivityappearsdistinctfromthatofChTXsinceIbTXdecreasesboththeprobABIlityofchannelopeningaswellasthechannelmeanopentime.IbTXisaselectiveinhibitorofPK,Ca;itdoesnotblockothertypesofvoltage-dependentionchannels,especiallyothertypesofK+channelsthataresensitivetoinhibitionbyChTX.IbTXisapartialinhibitorof125I-ChTXbindinginbovineaorticsarcolemmalmembranevesicles(Ki=250pM).ThemaximalextentofinhibitionthatoccursismodulatedbyK+,decreasingasK+concentrationisraised,butK+doesnotaffecttheabsoluteinhibitorypotencyofIbTX.AScatchardanalysisindicatesthatIbTXfunctionsasanoncompetitiveinhibitorofChTXbinding.Takentogether,thesedatasuggestthatIbTXinteractsatadistinctsiteonthechannelandmodulatesChTXbindingbyanallostericmechanism.Therefore,IbTXdefinesanewclassofpeptidylinhibitorofPK,Cawithuniquepropertiesthatmakeitusefulforinvestigatingthecharacteristicsofthischannelintargettissues.
GalvezA,etal.Purificationandcharacterizationofaunique,potent,peptidylprobeforthehighconductancecalcium-activatedpotassiumchannelfromvenomofthescorpionButhustamulus.JBiolChem.PMID: 1694175
Mechanismofiberiotoxinblockofthelarge-conductancecalcium-activatedpotassiumchannelfrombovineaorticsmoothmuscle
Theinteractionofiberiotoxin(IbTX)withthelarge-conductancecalcium-activatedpotassium(maxi-K)channelwasexaminedbymeasuringsingle-channelcurrentsfrommaxi-Kchannelsincorporatedintoplanarlipidbilayers.AdditionofnanomolarconcentrationsofIbTXtotheexternalsideofthechannelproducedlongnonconductingsilentperiods,whichwereinterruptedbyperiodsofnormalchannelactivity.Thedistributionsofdurationsofblockedandunblockedperiodswerebothdescribedbysingleexponentials.ThemeandurationoftheunblockedperiodsdecreasedinproportionwiththeexternalconcentrationofIbTX,whilethemeandurationoftheblockedperiodswasnotaffected.TheseresultssuggestthatIbTXblocksthemaxi-Kchannelthroughasimplebimolecularbindingreactionwherethesilentperiodsrepresenttimeswhenasingletoxinmoleculeisboundtothechannel.Insymmetricsolutionsof150mMKCl,withamembranepotentialof40mV,themeandurationoftheblockedperiodsproducedbyIbTXwas840s,andtheassociationratewas1.3x10(6)M-1s-1,yieldinganequilibriumdissociationconstantofabout1nM.RaisingtheinternalpotassiumconcentrationincreasedthedissociationrateconstantofIbTXinamannerwhichwaswelldescribedbyasaturablebindingfunctionforpotassium.Externaltetraethylammoniumionincreasedtheaveragedurationoftheunblockedperiodswithoutaffectingtheblockedperiods,suggestingthattetraethylammoniumandIbTXcompeteforthesamesiteneartheconductancepathwayofthechannel.Increasingtheexternalconcentrationofmonovalentcationsfrom25to300mMwitheitherpotassiumorsodiumdecreasedtherateofbindingofIbTXtothechannelbyapproximately24-fold,withlittleeffectontherateoftoxindissociation.
GiangiacomoKM,etal.Mechanismofiberiotoxinblockofthelarge-conductancecalcium-activatedpotassiumchannelfrombovineaorticsmoothmuscle.Biochemistry.PMID: 1379069
High-conductancecalcium-activatedpotassiumchannels;structure,pharmacology,andfunction
High-conductancecalcium-activatedpotassium(maxi-K)channelscompriseaspecializedfamilyofK+channels.TheyareuniqueintheirdualrequirementfordepolarizationandCa2+bindingfortransitiontotheopen,orconducting,state.Ionconductionthroughmaxi-Kchannelsisblockedbyafamilyofvenom-derivedpeptides,suchascharybdotoxinandiberiotoxin.Thesepeptideshavebeenusedtostudyfunctionandstructureofmaxi-Kchannels,toidentifynovelchannelmodulators,andtofollowthepurificationoffunctionalmaxi-Kchannelsfromsmoothmuscle.Thechannelconsistsoftwodissimilarsubunits,alphaandbeta.ThealphasubunitisamemberofthesloCa(2+)-activatedK+channelgenefamilyandformstheionconductionpore.Thebetasubunitisastructurallyunique,membrane-spanningproteinthatcontributestochannelgatingandpharmacology.Potent,selectivemaxi-Kchanneleffectors(bothagonistsandblockers)oflowmolecularweighthavebeenidentifiedfromnaturalproductsources.Theseagents,togetherwithpeptidylinhibitorsandsite-directedantibodiesraisedagainstalphaandbetasubunitsequences,canbeusedtoanatomicallymapmaxi-Kchannelexpression,andtostudythephysiologicroleofmaxi-Kchannelsinvarioustissues.Onegoalofsuchinvestigationsistodeterminewhethermaxi-Kchannelsrepresentnoveltherapeutictargets.
KaczorowskiGJ,etal.High-conductancecalcium-activatedpotassiumchannels;structure,pharmacology,andfunction.JBioenergBiomembr. PMID: 8807400
Useoftoxinstostudypotassiumchannels
Potassiumchannelscomprisegroupsofdiverseproteinswhichcanbedistinguishedaccordingtoeachmember’sbiophysicalproperties.SometypesofK+channelsareblockedwithhighaffinitybyspecificpeptidyltoxins.Threetoxins,charybdotoxin,iberiotoxin,andnoxiustoxin,whichdisplayahighdegreeofhomologyintheirprimaryaminoacidsequences,havebeenpurifiedtohomogeneityfromscorpionvenom.Whilecharybdotoxinandnoxiustoxinareknowntoinhibitmorethanoneclassofchannel(i.e.,severalCa(2+)-activatedandvoltage-dependentK+channels),iberiotoxinappearstobeaselectiveblockerofthehigh-conductance,Ca(2+)-activatedK+channelthatispresentinmuscleandneuroendocrinetissue.Adistinctclassofsmall-conductanceCa(2+)-activatedK+channelisblockedbytwoothertoxins,apaminandleiurotoxin-1,thatsharenosequencehomologywitheachother.Afamilyofhomologoustoxins,thedendrotoxins,havebeenpurifiedfromvenomofvariousrelatedspeciesofsnakes.Thesetoxinsinhibitseveralinactivatingvoltage-dependentK+channels.Althoughmolecularbiologyapproacheshavebeenemployedtoidentifyandcharacterizeseveralspeciesofvoltage-gatedK+channels,toxinsdirectedagainstaparticularchannelcanstillbeusefulindefiningthephysiologicalroleofthatchannelinaparticulartissue.Inaddition,forthoseK+channelswhicharenotyetsuccessfullyprobedbymolecularbiologytechniques,toxinscanbeusedasbiochemicaltoolswithwhichtopurifythetargetproteinofinterest.
GarciaML,etal.Useoftoxinstostudypotassiumchannels.JBioenergBiomembr. PMID: 1917911
Modeofactionofiberiotoxin,apotentblockerofthelargeconductanceCa(2+)-activatedK+channel
Iberiotoxin,atoxinpurifiedfromthescorpionButhustamulusisa37aminoacidpeptidehaving68%homologywithcharybdotoxin.CharybdotoxinblockslargeconductanceCa(2+)-activatedK+channelsatnanomolarconcentrationsfromtheexternalsideonly(Miller,C.,E.Moczydlowski,R.Latorre,andM.Phillips.1985.Nature(Lond.).313:316-318).Likecharybdotoxin,iberiotoxinisonlyabletoblocktheskeletalmusclemembraneCa(2+)-activatedK+channelincorporatedintoneutral-planarbilayerswhenappliedtotheexternalside.Inthepresenceofiberiotoxin,channelactivityisinterruptedbyquiescentperiodsthatcanlastforseveralminutes.Fromsingle-channelrecordsitwaspossibletodeterminethatiberiotoxinbindstoCa(2+)-activateK+channelinabimolecularreaction.Whenthesolutionbathingthemembraneare300mMK+internaland300mMNa+externalthetoxinsecondorderassociationrateconstantis3.3x10(6)s-1M-1andthefirstorderdissociationrateconstantis3.8x10(-3)s-1,yieldinganapparentequilibriumdissociationconstantof1.16nM.Thisconstantis10-foldlowerthanthatofcharybdotoxin,andthevaluesfortherateconstantsshowedaboveindicatethatthisismainlyduetotheverylowdissociationrateconstant;meanblockedtimeapproximately5min.Thefactthattetraethylammoniumcompetitivelyinhibitstheiberiotoxinbindingtothechannelisastrongsuggestionthatthistoxinbindstothechannelexternalvestibule.IncreasingtheexternalK+concentrationmakestheassociationrateconstanttodecreasewithnoeffectonthedissociationreactionindicatingthatthesurfacechargeslocatedintheexternalchannelvestibuleplayanimportantroleinmodulatingtoxinbinding.
CandiaS.,etal.Modeofactionofiberiotoxin,apotentblockerofthelargeconductanceCa(2+)-activatedK+channel.BiophysJ. PMID: 1384740
