µ-conotoxinGIIIBisa22aminoacidconopeptideoriginallyisolatedfromthevenomofthepiscivorousmarinesnailConusgeographus.µ-conotoxinGIIIBadoptsacompactstructureconsistingofadistorted310-helixandasmallß-hairpin.µ-conotoxinGIIIBisstABIlizedbythreedisulphidebridgesandishighlyenrichedinlysineandarginineresidues,formingpotentialsitesofinteractionwithNachannels.Anunusualfeatureisthepresenceofthreehydroxyprolineresidues.µ-conotoxinGIIIBisausefulprobetodiscriminatebetweenneuronalandmusclesodiumchannelsasitexhibitsatleasta1000-foldspecificityformuscleversusnervesodiumchannels.µ-ConotoxinGIIIBselectivelyblocksNav1.4voltage-dependentsodiumchannels,whicharepredominantlyexpressedinmuscle,withanaffinitycloseto20nM.µ-ConotoxinGIIIBappearstophysicallyoccludethechannelporebybindingonsiteIoftheNa+channel.
Description:
AAsequence:Arg-Asp-Cys3-Cys4-Thr-Hyp-Hyp-Arg-Lys-Cys10-Lys-Asp-Arg-Arg-Cys15-Lys-Hyp-Met-Lys-Cys20-Cys21-Ala-NH2
Disulfidebonds:Cys3-Cys15,Cys4-Cys20andCys10-Cys21
Length(aa):22
Formula:C101H175N39O30S7
MolecularWeight:2640.26Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:
Source:Synthetic
Purityrate:>97%
Reference:
MolecularBasisofIsoform-specificμ-ConotoxinBlockofCardiac,SkeletalMuscle,andBrainNa+Channels
mu-Conotoxins(mu-CTXs)blockskeletalmuscleNa(+)channelswithanaffinity1-2ordersofmagnitudehigherthancardiacandbrainNa(+)channels.Althoughanumberofconservedporeresiduesarerecognizedascriticaldeterminantsofmu-CTXblock,themolecularbasisofisoform-specifictoxinsensitivityremainsunresolved.SequencecomparisonofthedomainII(DII)S5-S6loopsofratskeletalmuscle(mu1,Na(v)1.4),humanheart(hh1,Na(v)1.5),andratbrain(rb1,Na(v)1.1)Na(+)channelsrevealssubstantialdivergenceintheirN-terminalS5-PlinkerseventhoughtheP-S6andC-terminalPsegmentsarealmostidentical.WeusedNa(v)1.4asthebackboneandsystematicallyconvertedtheseDIIS5-PisoformvariantstothecorrespondingresiduesinNa(v)1.1andNa(v)1.5.TheNa(v)1.4–>Na(v)1.5variantsubstitutionsV724R,C725S,A728S,D730S,andC731S(Na(v)1.4numbering)reducedblockofNa(v)1.4by4-,86-,12-,185-,and55-foldrespectively,renderingtheskeletalmuscleisoformmore“cardiac-like.”Conversely,anNa(v)1.5–>Na(v)1.4chimericconstructinwhichtheNa(v)1.4DIIS5-PlinkerreplacestheanalogoussegmentinNa(v)1.5showedenhancedmu-CTXblock.However,thesevariantdeterminantsareconservedbetweenNa(v)1.1andNa(v)1.4andthuscannotexplaintheirdifferentsensitivitiestomu-CTX.ComparisonoftheirsequencesrevealstwovariantsatNa(v)1.4positions729and732:SerandAsninNa(v)1.4comparedwithThrandLysinNa(v)1.1,respectively.ThedoublemutationS729T/N732KrenderedNa(v)1.4more“brain-like”(30-folddownwardarrowinblock),andtheconversemutationT925S/K928NinNa(v)1.1reproducedthehighaffinityblockingphenotypeofNa(v)1.4.WeconcludethattheDIIS5-Plinker,althoughlyingoutsidetheconventionalion-conductingpore,playsaprominentroleinmu-CTXbinding,thusshapingisoform-specifictoxinsensitivity.
RonaldA.Li, etal.(2003)MolecularBasisofIsoform-specificμ-ConotoxinBlockofCardiac,SkeletalMuscle,andBrainNa+ Channels. JBC. PMID: 12471026
Conusgeographustoxinsthatdiscriminatebetweenneuronalandmusclesodiumchannels
Wedescribethepropertiesofafamilyof22-aminoacidpeptides,themu-conotoxins,whichareusefulprobesforinvestigatingvoltage-dependentsodiumchannelsofexcitabletissues.Themu-conotoxinsarepresentinthevenomofthepiscivorousmarinesnail,ConusgeographusL.Wehavepurifiedsevenhomologsofthemu-conotoxinsetanddeterminedtheiraminoacidsequences,asfollows,whereHyp=trans-4-hydroxyproline.GIIIAR.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2[Pro6]GIIIAR.D.C.C.T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2[Pro7]GIIIAR.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2GIIIBR.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2[Pro6]GIIIBR.D.C.C.T.P.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2[Pro7]GIIIBR.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2GIIICR.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2.Usingthemajorpeptide(GIIIA)inelectrophysiologicalstudiesonnerve-musclepreparationsandinsinglechannelstudiesusingplanarlipidbilayers,wehaveestablishedthatthetoxinblocksmusclesodiumchannels,whilehavingnodiscernIBLeeffectonnerveorbrainsodiumchannels.InbilayerstheblockingkineticsofGIIIAwerederivedbystatisticalanalysisofdiscretetransitionsbetweenblockedandunblockedstatesofbatrachotoxin-activatedsodiumchannelsfromratmuscle.Thekineticsconformtoasingle-site,reversiblebindingequilibriumwithavoltage-dependentbindingconstant.ThemeasuredvalueoftheequilibriumKDforGIIIAis100nMatOmV,decreasinge-fold/34mVofhyperpolarization.Thisvoltagedependenceofblockingissimilartothatoftetrodotoxinandsaxitoxinasmeasuredbythesametechnique.Thetissuespecificityandkineticcharacteristicssuggestthatthemu-conotoxinsmayserveasusefulligandstodistinguishsodiumchannelsubtypesindifferenttissues.
CruzLJ, etal. (1985)Conusgeographustoxinsthatdiscriminatebetweenneuronalandmusclesodiumchannels. JBC. PMID: 2410412
NovelStructuralDeterminantsofm-Conotoxin(GIIIB)BlockinRatSkeletalMuscle(m1)Na+Channels
mu-Conotoxin(mu-CTX)specificallyoccludestheporeofvoltage-dependentNa(+)channels.IntheratskeletalmuscleNa(+)channel(mu1),weexaminedthecontributionofchargedresiduesbetweenthePloopsandS6inallfourdomainstomu-CTXblock.ConversionofthenegativelychargeddomainII(DII)residuesAsp-762andGlu-765tocysteineincreasedtheIC(50)formu-CTXblockbyapproximately100-fold(wild-type=22.3+/-7.0nm;D762C=2558+/-250nm;E765C=2020+/-379nm).Restorationorreversalofchargebyexternalmodificationofthecysteine-substitutedchannelswithmethanethiosulfonatereagents(methanethiosulfonateethylsulfonate(MTSES)andmethanethiosulfonateethylammonium(MTSEA))didnotaffectmu-CTXblock(D762C:IC(50,MTSEA+)=2165.1+/-250nm;IC(50,MTSES-)=2753.5+/-456.9nm;E765C:IC(50,MTSEA+)=2200.1+/-550.3nm;IC(50,MTSES-)=3248.1+/-2011.9nm)comparedwiththeirunmodifiedcounterparts.Incontrast,thecharge-conservingmutationsD762E(IC(50)=21.9+/-4.3nm)andE765D(IC(50)=22.0+/-7.0nm)preservedwild-typeblockingbehavior,whereasthechargereversalmutantsD762K(IC(50)=4139.9+/-687.9nm)andE765K(IC(50)=4202.7+/-1088.0nm)destabilizedmu-CTXblockevenfurther,suggestingaprominentelectrostaticcomponentoftheinteractionsbetweentheseDIIresiduesandmu-CTX.Kineticanalysisofmu-CTXblockrevealsthatthechangesintoxinsensitivityarelargelyduetoacceleratedtoxindissociation(k(off))rateswithlittlechangesinassociation(k(on))rates.Weconcludethattheacidicresiduesatpositions762and765arekeydeterminantsofmu-CTXblock,primarilybyvirtueoftheirnegativecharge.TheinabilityofthebulkyMTSESorMTSEAsidechaintomodifymu-CTXsensitivityplacesstericconstraintsonthesitesoftoxininteraction.
RonaldA.Li, etal. (2000)NovelStructuralDeterminantsofm-Conotoxin(GIIIB)BlockinRatSkeletalMuscle(m1)Na+ Channels.JBC.PMID: 10859326
HyperpolarizedshiftsinthevoltagedependenceoffastinactivationofNav1.4andNav1.5inaratmodelofcriticalillnessmyopathy
Criticalillnessmyopathyisadisorderinwhichskeletalmusclebecomeselectricallyinexcitable.Wepreviouslydemonstratedthatashiftinthevoltagedependenceoffastinactivationofsodiumcurrentscontributestoinexcitabilityofaffectedfibresinananimalmodelofcriticalillnessmyopathyinwhichdenervatedratskeletalmuscleistreatedwithcorticosteroids(steroid-denervated;SD).InthecurrentstudyweexaminedwhetherexpressionofNav1.5contributestothealteredvoltagedependenceofsodiumchannelinactivationinSDmuscle.WeusedTTXandmu-conotoxinGIIIBtoselectivelyblockNav1.4inSDmuscleandfoundthatthelevelofNav1.5didnotcorrelatecloselywiththeshiftinfastinactivation.Surprisingly,wefoundthatthevoltagedependenceofinactivationofNav1.4wassimilartothatofNav1.5inskeletalmuscleinvivo.Inseverelyaffectedfibres,inactivationofbothNav1.4andNav1.5wasshiftedtowardshyperpolarizedpotentials.Weexaminedtheroleofdenervationandsteroidtreatmentintheshiftofthevoltagedependenceofinactivationandfoundthatbothdenervationandsteroidtreatmentcontributetotheshiftininactivation.OurresultssuggestthatmodulationofthevoltagedependenceofinactivationofbothNav1.4andNav1.5invivocontributestolossofelectricalexcitabilityinSDmuscle.
GregoryN.FilatovandMarkM.Rich(2004)HyperpolarizedshiftsinthevoltagedependenceoffastinactivationofNav1.4andNav1.5inaratmodelofcriticalillnessmyopathy. J.Physiol. PMID: 15254148
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