Muscarinictoxin7(MT7–m1-toxin1)hasbeenisolatedfromthevenomofthegreenmamba(DendroaspisAngusticeps).MT7potentlyblocksM1-subtypeofmuscarinicacetylcholinereceptorsatasubnanomolaraffintiy.MuscarinicacetylcholinereceptorsareG-proteincoupledreceptorsthatmediatethemetabotropiceffectsofacetylcholine.M1-typemuscarinicacetylcholinereceptorsarewellknowntherapeutictargetstoimprovecognitivefunctionsinpatientswithAlzheimerdisease.ContrarytomanyligandsthattargetmAChRs(pirenzepine,carbamoylcholinechloride,4-DAMPoratropine),MT7isthemostselectiveM1-subtypeantagoNISTasitisabout10,000timesmoreselectiveforM1-subtypethanothersubtypes.MT7toxinisanidealtooltoidentifythemuscarinicreceptorsubtypeexpressionintissuesforexample.MT7toxinbelongstothethreefingertoxinfamilysuchasrho-Da1a.
Recentlyquoted
Description:
AAsequence:LTC3VKSNSIWFPTSEDC17PDGQNLC24FKRWQYISPRMYDFTRGC42AATC46PKAEYRDVINC57C58GTDKC63NK
Disulfidebonds:Cys3-Cys24,Cys17-Cys42,Cys46-Cys57,Cys58-Cys63
Length(aa):65
Formula:C322H484N90O98S9
MolecularWeight: 7472.53Da
Appearance:Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: notavailable
Source:synthetic
Purityrate: >98%
Reference:
Structuraldeterminantsfortheinteractionsbetweenmuscarinictoxin7andmuscarinicacetylcholinereceptors
XuJ.,etal.(2015)Structuraldeterminantsfortheinteractionsbetweenmuscarinictoxin7andmuscarinicacetylcholinereceptors.JMolRecognit.PMID:25683330
Muscarinicacetylcholinereceptors(mAChRs)havefivesubtypesandplaycrucialrolesinvariousphysiologicalfunctionsandpathophysiologicalprocesses.PoorsubtypespecificityofmAChRmodulatorshasbeenanobstacletodiscovernewtherapeuticagents.Muscarinictoxin7(MT7)isanaturalpeptidetoxinwithhighselectivityfortheM1receptor.Withthreetofiveresiduessubstituted,M3,M4,andM5receptormutantscouldbindtoMT7atnanomolarconcentrationastheM1receptor.However,thestructuralmechanismsexplainingMT7-mAChRsbindingarestilllargelyunknown.Inthisstudy,weconstructed10complexmodelsofMT7andeachmAChRsubtypeoritsmutant,performedmoleculardynamicssimulations,andcalculatedthebindingenergiestoinvestigatethemechanisms.OurresultssuggestedthatthestructuraldeterminantsfortheinteractionsonmAChRswerecomposedofsomecriticalresidueslocatedseparatelyintheextracellularloopsofmAChRs,suchasGlu4.56,Leu4.60,Glu/Gln4.63,Tyr4.65,Glu/Asp6.67,andTrp7.35.ThesubtypespecificityofMT7wasattributedtothenon-conservedresiduesatpositions4.56and6.67.ThesestructuralmechanismscouldfacilitatethediscoveryofnovelmAChRmodulatorswithhighsubtypespecificityandenhancetheunderstandingoftheinteractionsbetweenligandsandG-protein-coupledreceptors.
MolecularconversionofmuscarinicAcetylcholineReceptorM5toMuscarinicToxin(MT7)bindingprotein
RondinelliS.,etal.(2011)MolecularconversionofmuscarinicAcetylcholineReceptorM5toMuscarinicToxin(MT7)bindingprotein.Toxins.PMID:22174976
Muscarinictoxin7(MT7)isamambavenompeptidethatbindsselectivelytotheM(1)muscarinicacetylcholinereceptor.Wehavepreviouslyshownthatthesecond(ECL2)andthird(ECL3)extracellularloopsoftheM(1)receptorarecriticallyinvolvedinbindingthepeptide.InthisstudyweusedamutagenesisapproachontheM(5)subtypeofthereceptorfamilytofindoutifthispossessesasimilarstructuralarchitectureintermsoftoxinbindingastheM(1)receptor.AnM(5)receptorconstruct(M(5)-E(175)Y(184)E(474)),mutatedattheformerlydecipheredcriticalresiduesonECL2and3,gainedtheABIlitytobindMT7,butwithratherlowaffinityasdeterminedinafunctionalassay(apparentK(i)=24nM;apparentK(i)forM(1)=0.5nM).Afterscreeningfordifferentdomainsandresidues,wefoundaspecificresidue(P(179)toLinM(5))inthemiddleportionofECL2thatwasnecessaryforhighaffinitybindingofMT7(M(5)-EL(179)YE,apparentK(i)=0.5nM).MutationofP(179)toAconfirmedarolefortheleucinesidechaininthebindingofMT7.TogethertheresultsrevealnewbindinginteractionsbetweenreceptorsandtheMT7peptideandstrengthenthehypothesisthatECL2sequenceisofutmostimportanceforMTbindingtomuscarinicreceptors.
DifferentinteractionbetweenMT7toxinandthehumanmuscarinicM1ReceptorinitsfreesanN-Methylscopolamineoccupiedstates.
Fruchart-GaillardC.,etal.(2008)DifferentinteractionbetweenMT7toxinandthehumanmuscarinicM1ReceptorinitsfreesanN-Methylscopolamineoccupiedstates. MolPharmacol.PMID:18784346
MuscarinicMT7toxinisahighlyselectiveandpotentantagonistoftheM(1)subtypeofmuscarinicreceptorandactsbybindingtoanallostericsite.ToidentifythemoleculardeterminantsbywhichMT7toxininteractswiththisreceptorinitsfreeandNMS-occupiedstates,theeffectontoxinpotencyofalaninesubstitutionwasevaluatedinequilibriumandkineticbindingexperimentsaswellasinfunctionalassays.ThedeterminationofthecrystallographicstructureofanMT7-derivative(MT7-diiodoTyr51)allowedtheselectionofcandidateresiduesthatareaccessIBLeandpresentonbothfacesofthethreetoxinloops.TheequilibriumbindingdataareconsistentwithnegativecooperativitybetweenN-methylscopolamine(NMS)andwild-typeormodifiedMT7andhighlightthecriticalroleofthetipofthecentralloopofthetoxin(Arg34,Met35Tyr36)initsinteractionwiththeunoccupiedreceptor.Examinationofthepotencyofwild-typeandmodifiedtoxinstoallostericallydecreasethedissociationrateof[(3)H]NMSallowedtheidentificationoftheMT7residuesinvolvedinitsinteractionwiththeNMS-occupiedreceptor.Incontrasttotheresultswiththeunoccupiedreceptor,themostimportantresidueforthisinteractionwasTyr36inloopII,assistedbyTrp10inloopIandArg52inloopIII.ThecriticalroleofthetipsoftheMT7loopswasalsoconfirmedinfunctionalexperiments.ThehighspecificityoftheMT7-M(1)receptorinteractionexploitsseveralMT7-specificresiduesandrevealsadifferentmodeofinteractionofthetoxinwiththefreeandNMS-occupiedstatesofthereceptor.
MuscarinicToxin7selectivityisdictatedbyextracellularreceptorloops
KukkonenA.,etal.(2004)MuscarinicToxin7selectivityisdictatedbyextracellularreceptorloops.JBC.PMID: 15452105
Muscarinictoxin7(MT7)isamambavenomproteinantagonistwithextremelyhighselectivityfortheM1muscarinicacetylcholinereceptor.TomapthesitesfortheinteractionofMT7withmuscarinicreceptorswehaveusedchimericM1:M3receptorsandsite-directedmutagenesisoftheM3andM4receptorsubtypes.TwoGluresiduesinM1,oneinextracellularloop2andoneinextracellularloop3,werefoundtobeimportantforthehighaffinitybindingofMT7.SubstitutionofthecorrespondingLysresiduesintheM3receptorwithGluconvertedtheM3mutanttoanMT7bindingreceptor,albeitwithloweraffinitycomparedwithM1.APhe–>Tyrsubstitutioninextracellularloop2ofM3togetherwiththe2GlumutationsgeneratedareceptorwithanincreasedMT7affinity(apparentKi=0.26nMinafunctionalassay)comparedwiththeM1receptor(apparentKi=1.31nM).TheimportanceoftheidentifiedaminoacidresidueswasconfirmedwithamutatedM4receptorconstructs.TheresultsindicatethatthehighselectivityofMT7fortheM1receptordependsonveryfewresidues,thusprovidinggoodProspectsforfuturedesignandsynthesisofmuscarinicreceptor-selectiveligands.
EffectsofmuscarinictoxinsMT2andMT7,fromgreenmambavenom,onm1,m3andm5muscarinicreceptorsexpressedinChineseHamsterOvarycells
BradleyKN.etal.(2003)EffectsofmuscarinictoxinsMT2andMT7,fromgreenmambavenom,onm1,m3andm5muscarinicreceptorsexpressedinChineseHamsterOvarycells.Toxicon.PMID:12565740
Severalsmallproteinscalledmuscarinictoxins(MTs)havebeenisolatedfromvenomofgreenmamba(Dendroaspisangusticeps).TheyhavepreviouslybeenshowninrADIoligandbindingstudiestohavehighselectivityandaffinityforindividualmuscarinicreceptorsubtypes,butlessisknownoftheirfunctionaleffects.ThisstudyhasexaminedtheactionsoftwooftheseMTs,MT2andMT7,usingchangesincytosolicCa(2+)([Ca(2+)](i))measuredusingthefluorescentindicatorfura-2inChineseHamsterOvary(CHO)cellsstablytransfectedwithindividualmuscarinicreceptorsubtypes,m1,m3andm5.MT2activatedthem1receptor:atconcentrationsabove100nMitcausedsignificantandconcentration-dependentincreasesin[Ca(2+)](i).From25to800nMMT2alsoproducedincreasesin[Ca(2+)](i)byactivatingm3receptors,althoughtheseincreasesin[Ca(2+)](i)werenotstrictlyconcentration-dependentwithonlyintermittentresponsesbeingrecorded(i.e.itwasnotalwayspossibletoobtainaresponsetotheagonistwitheachapplicationofthecompound).MT2(800-1600nM)alsocausedsignificantincreasesin[Ca(2+)](i)inCHOcellsexpressingthem5muscarinicreceptorsubtype.MT7(1microM)displayednoagonistactivityatanyofthemuscarinicreceptorsbutwasapotentnon-competitiveantagonist(at20nM)atthem1muscarinicreceptorsubtype.Ithadnoantagonistactivityatthem3orm5subtypes.TheseresultsindicatethatMT7isahighlyspecificantagonistatthem1muscarinicreceptorsubtypeassuggestedbyresultsfromradioligandbindingstudies.However,MT2islessselectiveforthem1muscarinicreceptorthanpreviouslydescribedasitalsoexhibitsagonistactivityatthem3andm5muscarinicreceptors,whichwasnotdetectedinradioligandbindingstudies.
InhibitionofacetylcholinemuscarinicM1receptorfunctionbytheM1-selectiveligandmuscarinictoxin7(MT-7)
OlianasMC.,etal.(2000)InhibitionofacetylcholinemuscarinicM1receptorfunctionbytheM1-selectiveligandmuscarinictoxin7(MT-7).BrJPharmacol.PMID:11015294
MT-7(1–30nM),apeptidetoxinisolatedfromthevenomofthegreenmambaDendroaspisangusticepsandpreviouslyfoundtobindselectivelytothemuscarinicM(1)receptor,inhibitedtheacetylcholine(ACh)-stimulated[(35)S]-guanosine-5′-O-(3-thio)triphosphate([(35)S]-GTPgammaS)bindingtomembranesofChinesehamsterovary(CHO)cellsstablyexpressingtheclonedhumanmuscarinicM(1)receptorsubtype.MT-7failedtoaffecttheACh-stimulated[(35)S]-GTPgammaSbindinginmembranesofCHOcellsexpressingeithertheM(2),M(3)orM(4)receptorsubtype.InN1E-115neuroblastomacellsendogenouslyexpressingtheM(1)andM(4)receptorsubtypes,MT-7(0.3–3.0nM)inhibitedthecarbachol(CCh)-stimulatedinositolphosphatesaccumulation,butfailedtoaffecttheCCh-inducedinhibitionofpituitaryadenylatecyclaseactivatingpolypeptide(PACAP)38-stimulatedcyclicAMPaccumulation.InbothCHO/M(1)andN1E-115cellstheMT-7inhibitionconsistedinadecreaseofthemaximalagonisteffectwithminimalchangesintheagonistEC(50)value.InCHO/M(1)cellmembranes,MT-7(0.05–25nM)reducedthespecificbindingof0.05,1.0and15nM[(3)H]-N-methylscopolamine([(3)H]-NMS)inaconcentration-dependentmanner,butfailedtocauseacompletedisplacementoftheradioligand.Moreover,MT-7(3nM)decreasedthedissociationrateof[(3)H]-NMSbyabout5fold.CHO/M(1)cellmembranespreincubatedwithMT-7(10nM)andwashedbycentrifugationandresUSPensiondidnotrecovercontrol[(3)H]-NMSbindingforatleast8hat30degreesC.ItisconcludedthatMT-7actsasaselectivenoncompetitiveantagonistofthemuscarinicM(1)receptorsbybindingstablytoanallostericsite.
