Conantokin-GisaconopeptidethathasbeenisolatedfromthevenomoftheconeConusgeographus. Conantokin-Gselectivelyinhibits NR2B containingN-methyl-D-aspartatereceptors(NMDAR)withahighspecificity.Thus, Conantokin-G inhibitsinaconcentration-dependentmannertheCa2+ influxleADIngtoaneuroprotectiveeffectagainstNMDA-inducedexcitotoxicity. Conantokin-G wasshowntoblockNMDA-evokedcurrentsinmurinecorticalneuronswithanIC50 valueofaround480nM.
Description:
AAsequence: Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2
Length(aa): 17
Formula: C88H138N26O44
MolecularWeight: 2264.23Da
Appearance: Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: [93438-65-4]Source: Synthetic
Purityrate: >95%
Reference:
Opposingactionofconantokin-Gonsynapticallyandextrasynaptically-activatedNMDAreceptors
SynapticandextrasynapticactivationoftheN-methyl-D-aspartatereceptor(NMDAR)hasdistinctconsequencesoncellsignalingandneuronalsurvival.Sinceconantokin(con)-GantagonismisNR2B-selective,whichisthekeysubunitinvolvedinextrasynapticactivationofthereceptor,itsABIlitytospecificallyelicitdistinctsignalingoutcomesinneuronswithsynapticallyorextrasynaptically-activatedNMDARswasevaluated.InhibitionofCa(2+)influxthroughextrasynapticNMDARionchannelswasneuroprotective,asiteffectivelyenhancedlevelsofactivatedextracellularsignal-regulatedkinase1/2(ERK1/2),activatedcAMPresponseelementbindingprotein(CREB),enhancedmitochondrialviability,andattenuatedtheactindisorganizationobservedbyextrasynapticactivationofNMDARs.Conversely,thepro-signalingpathwaysstimulatedbysynaptically-inducedCa(2+)influxwereabolishedbycon-G.FurThermore,subunitnon-selectivecon-Twasunabletosuccessfullyredresstheimpairmentsinneuronscausedbyextrasynaptically-activatedNMDARs,thusindicatingthatNR2B-specificantagoNISTsarebeneficialforneuronsurvival.NeuronsablatedfortheNR2BsubunitshowedweaksynapticCa(2+)influx,reducedsensitivitytoMK-801blockage,anddiminishedextrasynapticcurrentcomparedtoWTandNR2A(-/-)neurons.ThisindicatesthattheNR2BsubunitisanintegralcomponentofbothsynapticandextrasynapticNMDARchannels.Altogether,thesedatasuggestthatcon-GspecificallytargetstheNR2Bsubunitinthesynapticandextrasynapticlocations,resultingintheopposingactionofcon-GondifferentiallyactivatedpoolsofNMDARs.
BalsaraR., etal. (2012)Opposingactionofconantokin-Gonsynapticallyandextrasynaptically-activatedNMDAreceptors.Neuropharmacology. PMID: 22306487
CGX-1007preventsexcitotoxiccelldeathviaactionsatmultipletypesofNMDAreceptors
Glutamateinducedexcitotoxicinjurythroughover-activationofN-methyl-D-aspartatereceptors(NMDARs)playsacriticalroleinthedevelopmentofmanyneurodegenerativediseases.ThepresentstudywasundertakentoevaluatetheroleofCGX-1007(ConantokinG)asaneuroprotectiveagentagainstNMDA-inducedexcitotoxicity.ConantokinG,aconesnailpeptideisolatedfromConusgeographusisreportedtoselectivelyinhibitNR2BcontainingNMDARswithhighspecificityandisshowntohavepotentanticonvulsantandantinociceptiveeffects.CGX-1007significantlyreducedtheexcitotoxiccelldeathinducedbyNMDAinorganotypichippocampalbrainsliceculturesinaconcentration-dependentmanner.Incontrast,ifenprodil,anotherNR2BspecificantagonistfailedtoofferneuroprotectionagainstNMDA-inducedexcitotoxicity.WefurtherdeterminedthattheneuroprotectionobservedislikelyduetotheactionofCGX-1007atmultipleNMDAreceptorsubtypes.Inaseriesofelectrophysiologyexperiments,CGX-1007inhibitedNMDA-gatedcurrentsinhumanembryonickidney(HEK)293cellsexpressingNMDAreceptorscontainingeitherNR1a/NR2BorNR1a/NR2Asubunitcombinations.CGX-1007producedaweakinhibitionatNR1a/NR2Creceptors,whereasithadnoeffectonNR1a/NR2Dreceptors.Further,theinhibitionofNMDAreceptorsbyCGX-1007wasvoltage-dependentwithgreaterinhibitionseenathyperpolarizedmembranepotentials.Thevoltage-dependenceofCGX-1007activitywasalsoobservedinrecordingsofNMDA-gatedcurrentsevokedinnativereceptorsexpressedincorticalneuronsinculture.Basedonourresults,weconcludethatCGX-1007isapotentneuroprotectiveagentthatactsasanantagonistatbothNR2AandNR2Bcontainingreceptors.
AlexAB., etal. (2011)CGX-1007preventsexcitotoxiccelldeathviaactionsatmultipletypesofNMDAreceptors.Neurotoxicology. PMID: 21396956
SpecificdeterminantsofconantokinsthatdictatetheirselectivityfortheNR2BsubunitofN-methyl-D-aspartatereceptors.
Conantokinsarenaturally-occurringsmallpeptideantagonistsofionflowthroughNMDA/glycineactivated-N-methyl-d-aspartatereceptor(NMDAR)ionchannels.Onememberoftheconantokinfamily,conantokin(con)-G,a17-residuepeptide,isselectiveforNMDARscontainingtheN-methyl-d-aspartatereceptorsubunit2B(NR2B),whereasthehomologouspeptides,con-Tandcon-R,showbroaderselectivityforNR2subunits.Inthisstudy,con-G,con-R,andcon-Tvariantswerechemicallysynthesizedandemployedtoinvestigatetheirsubunitselectivitiesasinhibitorsofagonist-evokedioncurrentsinhumanembryonickidney-293(HEK-293)cellsexpressingvariouscombinationsofNMDARsubunitsthatcontainNR1aorNR1bcombinedwithNR2AorNR2B.Usingtruncationandpointmutants,aswellaschimericconantokins,wedeterminedthattheN-terminusofcon-GcontainsallthedeterminantsforNR2Bselectivity.Withthisinformation,alargenumberof(con)variantsweresynthesizedandusedtoestablishminimalsequencedeterminantsforselectivity.Tyratposition5broadenstheNR2selectivity,andrecoveryofNR2BselectivityinTyr5peptideswasachievedbyincorporatingAlaorGlyatposition8.NR2Bselectivityincon-RcanbeconferredthroughdeletionoftheAlaatposition10,therebyshiftingtheγ-carboxyglutamate(Gla)fromposition11toposition10,whereaGlanaturallyoccursincon-Gandcon-T.Thenatureoftheaminoacidatposition6isalsolinkedtosubunitselectivity.OurstudiessuggestthatthemoleculardeterminantsofconantokinsthatdictateNMDARsubunitselectivityarehousedinspecificresiduesoftheN-terminiofthesepeptides.Thus,itispossIBLetoengineerdesiredNMDARfunctionalpropertiesintoconantokin-basedpeptides.
ShengZ., etal. (2010)SpecificdeterminantsofconantokinsthatdictatetheirselectivityfortheNR2BsubunitofN-methyl-D-aspartatereceptors. Neuroscience. PMID: 20688135
Theselectivityofconantokin-GforionchannelinhibitionofNR2Bsubunit-containingNMDAreceptorsisregulatedbyaminoacidresiduesintheS2regionofNR2B
Theconantokinsareshort,naturallyoccurringpeptidesthatinhibitionflowthroughN-methyl-d-aspartatereceptor(NMDAR)channels.Onememberofthispeptidefamily,conantokin-G(con-G),showshighselectivityforantagonismofNR2B-containingNMDARchannels,whereasotherknownconantokinsarelessselectiveinhibitorswithregardtothenatureoftheNR2subunitoftheNMDARcomplex.InordertodefinethemoleculardeterminantsofNR2Bthatgoverncon-Gselectivity,weevaluatedtheabilityofcon-GtoinhibitNMDARionchannelsexpressedinhumanembryonickidney(HEK)293cellstransfectedwithNR1,incombinationwithvariousNR2A/2Bchimerasandpointmutants,byelectrophysiologyusingcellsvoltage-clampedinthewhole-cellconfiguration.Wefoundthatavariantofthecon-G-insensitivesubunit,NR2A,inwhichthe158residuescomprisingtheS2peptidesegment(E(657)-I(814))werereplacedbythecorrespondingS2regionofNR2B(E(658)-I(815)),resultsinreceptorsthatarehighlysensitivetoinhibitionbycon-G.Ofthe22aminoacidsthataredifferentbetweentheNR2A-S2andtheNR2B-S2regions,exchangeofoneofthese,M(739)ofNR2BfortheequivalentK(738)ofNR2A,wassufficienttocompletelyimporttheinhibitoryactivityofcon-GintoNR1b/NR2A-containingNMDARs.Somereinforcementofthiseffectwasfoundbysubstitutionofasecondaminoacid,K(755)ofNR2BforY(754)ofNR2A.ThediscoveryofthemoleculardeterminantsofNR2Bselectivitywithcon-Ghasimplicationsforthedesignofsubunit-selectiveneuroBIOLOGicalprobesanddrugtherapies,inadditiontoadvancingourunderstandingofNR2B-versusNR2A-mediatedneurologicalprocesses.
ShengZ., etal.(2009)Theselectivityofconantokin-GforionchannelinhibitionofNR2Bsubunit-containingNMDAreceptorsisregulatedbyaminoacidresiduesintheS2regionofNR2B. Neuropharmacology. PMID: 19427876
Powerfulantinociceptiveeffectsoftheconesnailvenom-derivedsubtype-selectiveNMDAreceptorantagonistsconantokinsGandT
Subunitnon-selectiveN-methyl-D-aspartate(NMDA)receptorantagonistsreduceinjury-inducedpainbehavior,butgenerallyproduceunacceptablesideeffects.Inthisstudy,weexaminedtheantinociceptiveandmotoreffectsofconesnailvenom-derivedpeptides,conantokinsGandT(conGandconT),whichareselectiveinhibitorsoftheNR2BorNR2AandNR2BsubtypesoftheNMDAreceptor,respectively.WetestedtheeffectsofconGandconTinmodelsoftissue(formalintest),nerveinjury(partialsciaticnerveligation)andinflammation-induced(intraplantarCompleteFreund’sAdjuvant;CFA)paininmice.Intheformalintest,intrathecal(i.t.)conGorconTsuppressedtheongoingpainbehavior(ED(50)and95%confidenceintervals(CI),11(7-19)and19(11-33),respectively)atdosesthatwere17-27timeslowerthanthoserequiredtoimpairmotorfunction(acceleratingrotarodtreadmilltest:ED(50)and95%CI,300(120-730)and320(190-540)pmol,respectively).Bycomparison,SNX-111,anN-typevoltage-sensitivecalciumchannelantagonistthatisalsoderivedfromconesnailvenom,producedsignificantmotorimpairmentatadose(3.0pmol,i.t.)thatwasonlypartiallyefficaciousintheformalintest.Furthermore,conGreversedtheallodyniaproducedbynerveinjury,withgreaterpotencyonthermal(ED50and95%CI,24(10-55)pmol)thanonmechanicalallodynia(59(33-105)pmol).Finally,asingledoseofconG(100pmol,i.t.)alsoreducedCFA-evokedthermalandmechanicalallodynia.Takentogether,theseresultsdemonstratethatconantokinsexhibitpotentantinociceptiveeffectsinseveralmodelsofinjury-inducedpain.ThestudysupportsthenotionthatdrugsdirectedagainstsubtypesoftheNMDAreceptor,byvirtueoftheirreducedside-effectprofile,holdpromiseasnoveltherapeuticagentsforthecontrolofpain.
Malmberg,AB., etal.(2003)Powerfulantinociceptiveeffectsoftheconesnailvenom-derivedsubtype-selectiveNMDAreceptorantagonistsconantokinsGandT, Pain. PMID: 12507705
Theaminoacidresidueatsequenceposition5intheconantokinpeptidespartiallygovernssubunit-selectiveantagonismofrecombinantN-methyl-D-aspartatereceptors
Wholecellvoltageclamprecordingswereperformedtoassesstheabilityof conantokin-G(con-G), conantokin-T(con-T),anda17-residue truncatedformof conantokin-R(con-R[1-17])toinhibit N-methyl-d-aspartate (NMDA)-evokedcurrentsinhumanembryonickidney293cellstransientlyexpressingvariouscombinationsofNR1a,NR1b,NR2A,andNR2Breceptorsubunits.Con-Tandcon-R[1-17]attenuatedioncurrentsincellsexpressingNR1a/NR2AorNR1a/NR2B.Con-GdidnotaffectNMDA-evokedioniccurrentsincellsexpressingNR1a/NR2A,butitshowedinhibitoryactivityincellsexpressingNR1a/NR2B receptors andthetriheteromericcombinationofNR1a/NR2A/NR2B.AnAla-richcon-Ganalog,con-G[Q6G/gamma7K/N8A/gamma10A/gamma14A/K15A/S16A/N17A](Ala/con-G,wheregammaisGla),inwhichallnonessential amino acidswerealteredtoAlaresidues,manifestedsubunitspecificitysimilartothatofcon-G,suggestingthatthereplacedresiduesarenotresponsibleforselectivityinthecon-Gframework.Asarcosine-containingcon-Ttruncationanalog,con-T[1-9/G1Src/Q6G],inhibitedcurrentsinNR1a/NR2AandNR1a/NR2Breceptors,eliminatingresidues10-21asmediatorsofthebroadsubunitselectivityofcon-T.Incontrasttothenulleffectsofcon-GandAla/con-GataNR1a/NR2A-containingreceptor,someinhibition(approximately40%)ofNMDA-evokedcurrentswaseffectedbythese peptides incellsexpressingNR1b/NR2A.Thisfindingsuggeststhatthepresenceofexon 5 inNR1bplaysaroleintheactivityoftheconantokins.Analysisofvarious conantokinanalogsdemonstratedthatLeu(5)ofcon-Gisanimportantdeterminantof conantokin selectivity.Takenasawhole,theseresultssuggestthattheimportantmoleculardeterminantsonconantokinsresponsibleforNMDAreceptoractivityandspecificityarediscretelyhousedinspecificresiduesofthese peptides,thusallowingmolecularmanipulationoftheNMDAreceptorinhibitorypropertiesoftheconantokins.
Klein,R.C.,etal.(2001)Theaminoacidresidueatsequenceposition5intheconantokinpeptidespartiallygovernssubunit-selectiveantagonismofrecombinantN-methyl-D-aspartatereceptors, JBiolChem.PMID: 11335724
ConantokinGisanNR2B-selectivecompetitiveantagonistofN-methyl-D-aspartatereceptors.
ConantokinG(ConG)isa17-amino-acidpeptideantagonistofN-methyl-D-aspartate(NMDA)receptorsisolatedfromthevenomofthemarineconesnail,Conusgeographus.ThemechanismofactionofConGhasnotbeenwelldefined;bothcompetitiveandnoncompetitiveinteractionswiththeNMDA-bindingsitehavebeenproposed.InthisstudythemechanismofactionandsubunitselectivityofConGwasexaminedinwhole-cellvoltage-clamprecordingsfromculturedneuronsandintwoelectrodevoltage-clamprecordingsfromXenopusoocytesexpressingrecombinantNMDAreceptors.ConGwasapotentandselectiveantagonistofNMDA-evokedcurrentsinmurinecorticalneurons(IC(50)=480nM).TheslowonsetofConGblockcouldbepreventedbycoapplicationwithhighconcentrationsofNMDAorofthecompetitiveantagonist(RS)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonicacid.Furthermore,inoocytesexpressingNR1a/NR2Breceptors,ConGproducedarightwardshiftintheconcentration-responsecurveforNMDA,providingsupportforacompetitiveinteractionwiththeNMDA-bindingsite.ConGproducedanapparentnoncompetitiveshiftintheconcentration-responsecurveforsperminepotentiationofNMDAresponses,butthiswasduetospermine-inducedenhancementofConGblock.SpermineproducedasimilarenhancementofDL-2-amino-S-phosphopentanoicacidblock.Finally,ConGselectivelyblockedNMDAreceptorscontainingtheNR2Bsubunit.TheseresultsdemonstratethatConGisasubunit-specificcompetitiveantagonistofNMDAreceptors.TheuniquesubunitselectivityprofileofConGmayexplainitsfavorableinvivoprofilecomparedwithnonselectiveNMDAantagonists.
DonevanSD., etal. (2000)ConantokinGisanNR2B-selectivecompetitiveantagonistofN-methyl-D-aspartatereceptors. MolPharmacol. PMID: 10953056
Bindingofcationstoindividualgamma-carboxyglutamateresiduesofconantokin-Gandconantokin-T
Conantokin-G(con-G)andconantokin-T(con-T)arenaturallyoccurringgamma-carboxyglutamate(Gla)-containingpeptidesthatinteractwithmultivalentcationsinfunctionallyrelevantmanners.Selective13C-enrichmentofCgammaandCdeltaineachoftheGlaresidueshasallowedmetalbindingaffinitiestobemeasuredatindividualsidechains.Con-Tpossessestwometalbindingsites,onewithhighaffinityatGla10/Gla14andanotherwithweakbindingatGla3/Gla4.Con-GcontainstwositesofcomparablelowaffinityforCa2+.Analysisofthe13Cline-widthsofcon-GinthepresenceofMg2+allowedtheorderofmetalbindingtobedetermined,withGla10/Gla14loadingbeforetheGla3/Gla4/Gla7cluster.Whilethevariantpeptide,apo-con-T[Lys7Gla],wasshowntohaveaverylowalpha-helicalcontent,thispeptidebindsasecondmetalwithmuchgreateraffinitythanwild-typecon-T.ThisprovidesadditionalevidencethatGla7incon-Gisprimarilyresponsiblefordestabilizingtheapo-form,butisanimportantligandformetalchelation.Theresidue-specificalpha-helicalstabilitiesofcon-Gandcon-Tintheirmetal-freeandmetal-loadedstateswereestimatedbydeterminingratesofprotonexchangefrombackbonepeptidebondamideswithdeuteriumatomsfrom2H20-containingsolvents.Forbothpeptides,thelifetimesofprotonsonseveralpeptidebondamidesincreasedasmetalsofhigheraffinitywereboundtothepeptides,withthelongesthalf-livesfoundintheregionofthealpha-helicalturnstabilizedbytheGla10/Gla14metalcoordinationsite.WeproposethatGla10andGla14constitutetheprimarytightmetalionbindingsiteinbothpeptides.Thisdetailedanalysiswithphysiologicallyrelevantmetalcationsiscrucialfordecipheringtherolesofcriticalaminoacidsinthebioactivityoftheconantokinpeptides.
Blandl,T., etal.(1999)Bindingofcationstoindividualgamma-carboxyglutamateresiduesofconantokin-Gandconantokin-T, JPeptRes. PMID: 10406223
NMDA-receptorantagonistrequirementsinconantokin-G.
Aseriesofvariantsoftheneuroactive17-residuegamma-carboxyglutamate-(Gla)-containingpolypeptide,conantokin-G(con-G),weresynthesizedwiththeintentionofdeterminingthosefeaturesthatwereimportantforitsN-methyl-D-aspartate(NMDA)receptor-targetedantagonistactivityandforadoptionofitsdivalentcation-dependentalpha-helicalconformation.Employingthebindingof[3H]dizolcipine(MK-801)asanassayforopenreceptorionchannelsinratbrainmembranes,whichdisplaysinhibitionbycon-G(IC50=0.48microM),itwasfoundthatreplacementbyanAlaresidueofGla4ledtocompleteinactivationofthepeptide,whereasasimilarreplacementofGla3resultedina20-folddecreasedpotency.AlasubstitutionsforGla10andGla14didnotsubstantiallyaffect[3H]MK-801binding.ThissamesubstitutionatGla7appearedtoslightlyenhancebinding.Alareplacementsofnon-Glaresiduesdemonstratedthatfourofthem,viz.Glu2,Leu5,Gln9,andIle12,possessedatleast200-folddecreasesininhibitorypotency,whereassimilarreplacementsatGly1,Leu11,andArg13resultedinpeptideswith8-to12-foldincreasesintheIC50values.TheremainingaminoacidresiduestestedinthesingleAlareplacementseriesshowednosignificantchangesintheinhibitorycharacteristicsofwild-typecon-G.Additionalstudieswithcarboxyl-terminaltruncatedpeptidesrevealedthatthecarboxyl-terminal4aminoacidswereunimportantforthisactivity.Therewasnostrictcorrelationofinhibitionof[3H]MK-801bindingwiththeabilityofthesepeptidestoformcation-dependentalpha-helices.Peptideswithnotablylowalpha-helicalcontentinthepresenceofthesecationswerelackingatleastone,orboth,ofGla10andGla14.Con-G[Gla3,4,7,10,14E]andcon-G[Gla7,10,14E]weretheonlypeptidesthatremainedinacompletelyrandomconformationuponmetalionaddition.
BlandlT., etal. (1998)NMDA-receptorantagonistrequirementsinconantokin-G. FEBSLett. PMID: 9762921
Conantokin-G:anovelpeptideantagonisttotheN-methyl-D-asparticacid(NMDA)receptor
Conantokin-Gisa17aminoacidpeptideisolatedfromthevenomofthefish-eatingsnailConusgeographuswhichproduceshyperactivitywheninjectedintothebrainsofadultmice.WeshowthatthispeptideisaselectiveN-methyl-D-aspartate(NMDA)antagonistbasedonitsabilitytoblockNMDA-inducedelevationofcGMPinratcerebellarslicesinvitro(IC50=171nM),butnotkainicacid-inducedelevations.ThisinhibitioncouldnotbeovercomebyincreasingtheNMDAconcentration,indicatingnon-competitiveinhibition.Conantokin-Gdisplayednoaffinityforbindingsitesforthienylcyclohexylpiperidine,variousglutamatesubclassesorthoseforseveralotherneurotransmitters/neuromodulators.Thispeptide,however,enhanced[3H]glycinebindingtoratforebrainmembranesbutnottospinalcordmembranes.Theactivityprofileofthepeptideinvariousassaysindicatesthatitisanoveltypeofnon-competitiveNMDAantagonist.
MenaEE.,etal.(1990)Conantokin-G:anovelpeptideantagonisttotheN-methyl-D-asparticacid(NMDA)receptor.NeurosciLett. PMID: 2177176
