Tertiapin hasbeenisolatedfromthevenomoftheHoneybee Apismellifera. Tertiapin-Q isanoxidation-resistantmutantofthewild-typetertiapinwhereMethionine13hasbeenreplacedbyaGlutamine. Tertiapin-Q blockstheinwardlyrectifying Kir1.1(ROMK1) and Kir3.1/3.4(GIRK1/GIRK4alsoknownasIKACh) potassiumchannelswithKdvaluesofaround2nMand8nMrespectively.Tertiapin-Q alsoinhibitscalcium-activatedlargeconductance BKpotassiumchannels (KCa1.1)inaconcentrationandvoltage-dependentmanner(IC50 ~5nM),inadditiontoinhibiting Kir3.1/3.2(GIRK1/GIRK2) heteromultimerpotassiumchannelswithaKdcloseto270nM. Tertiapin-Q canpreventdose-dependentacetylcholine(ACh)-inducedatrioventricularblocksinmammalianhearts,asKCNJ3/KCNJ5channels(alsonamedI(KACh)),areactivatedbyAChfoundinmammalianmyocytes.
Description:
AAsequence: Ala-Leu-Cys3-Asn-Cys5-Asn-Arg-Ile-Ile-Ile-Pro-His-Gln-Cys14-Trp-Lys-Lys-Cys18-Gly-Lys-Lys-NH2
Disulfidebonds: Cys3-Cys14 andCys5-Cys18
Length(aa): 21
Formula: C106H175N35O24S4
MolecularWeight: 2452Da
Appearance:Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: [252198-49-5]Source: Synthetic
Purityrate: >97%
Reference:
CharacterizationofKir1.1channelswiththeuseofarADIolabeledderivativeoftertiapin,Biochemistry
Inwardrectifierpotassium channels (Kir)playcriticalrolesincellphysiology.Despiterepresentingthesimplesttetramericpotassiumchannelstructures,thepharmacologyofthischannelfamilyremainslargelyundeveloped.Inthisrespect, tertiapin (TPN),a21aminoacidpeptideisolatedfrombeevenom,hasbeenreportedtoinhibit Kir1.1 andKir3.1/3.4 channels withhighaffinitybybindingtotheM1-M2linkerregionofthese channels.Thefeaturesofthepeptide-channelinteractionhavebeenexploredelectrophysiologically,&thesestudieshaveidentifiedwaysbywhichtoalterthecompositionofthepeptidewithoutaffectingitsBIOLOGicalactivity.Inthepresentstudy,theTPN derivative,TPN-Y1/K12/Q13,hasbeensynthesized&radiolabeled tohighspecificactivitywith(125)I.TPN-Y1/K12/Q13&mono-iodo-TPN-Y1/K12/Q13([(127)I]TPN-Y1/K12/Q13)inhibitwithhighaffinityratbutnothuman Kir1.1 channels stablyexpressedinHEK293cells.[(125)I]TPN-Y1/K12/Q13bindsinasaturable,time-dependent,&reversIBLemannertoHEK293cellsexpressingrat Kir1.1,aswellastomembranesderivedfromthesecells,&thepharmacologyofthebindingreactionisconsistentwithpeptidebindingto Kir1.1 channels.Studiesusingchimeric channels indicatethatthedifferencesinTPNsensitivitybetweenrat&human Kir1.1 channels areduetothepresenceoftwononconservedresidueswithintheM1-M2linkerregion.Whentheseresultsaretakentogether,theydemonstratethat[(125)I]TPN-Y1/K12/Q13representsthefirsthighspecificactivityradioligandforstudyingrat Kir1.1 channels&suggestitsutilityforidentifyingotherKirchannelmodulators.
Felix,J.P.,etal.(2006)CharacterizationofKir1.1channelswiththeuseofaradiolabeledderivativeoftertiapin,Biochemistry. PMID: 16906771
Tertiapin-QblocksrecombinantandnativelargeconductanceK+channelsinause-dependentmanner
Tertiapin,ashortpeptidefromhoneybeevenom,hasbeenreportedtospecificallyblocktheinwardlyrectifying K(+)(Kir) channels,includingGprotein-coupledinwardlyrectifyingpotassiumchannel(GIRK)1+GIRK4heteromultimers&ROMK1homomultimers.Inthepresentstudy,theeffectsofastable&functionallysimilarderivativeoftertiapin, tertiapin-Q,wereexaminedon recombinant humanvoltage-dependentCa(2+)-activated largeconductance K(+)channel(BKorMaxiK;alpha-subunitorhSlo1homomultimers)&mouseinwardlyrectifyingGIRK1+GIRK2(i.e.,Kir3.1&Kir3.2)heteromultimeric K(+) channels expressedinXenopusoocytes&inculturednewbornmousedorsalrootganglion(DRG)neurons.Intwo-electrodevoltage-clampedoocytes, tertiapin-Q (1-100nM)inhibitedBK-type K(+) channels inause-&concentration-dependent manner.Wealsoconfirmedtheinhibitionof recombinant GIRK1+GIRK2heteromultimersby tertiapin-Q,whichhadnoeffectonendogenousdepolarization-&hyperpolarization-activatedcurrentssensitivetoextracellulardivalentcations(Ca(2+),Mg(2+),Zn(2+),&Ba(2+))indefolliculatedoocytes.Involtage-clampedDRGneurons, tertiapin-Q voltage-&use-dependentlyinhibitedoutwardlyrectifying K(+)currents,butCs(+)-blockedhyperpolarization-activatedinwardcurrentsincludingI(H)wereinsensitiveto tertiapin-Q,baclofen,barium,&zinc,suggestingabsenceoffunctionalGIRK channels inthenewborn.Undercurrent-clampconditions, tertiapin-Q blockedtheactionpotentialafterhyperpolarization(AHP)&increasedactionpotentialdurationinDRGneurons.Takentogether,theseresultsdemonstratethattheblockingactionsof tertiapin-Q arenotspecifictoKir channels&thattheblockadeofrecombinant BK channels&native neuronalAHPcurrentsis use-dependent.InhibitionofspecifictypesofKir&voltage-dependentCa(2+)-activated K(+) channels by tertiapin-Q atnanomolarrangeviadifferentmechanismsmayhaveimplicationsinpainphysiology&therapy.
Kanjhan,R.,C etal.(2005)Tertiapin-QblocksrecombinantandnativelargeconductanceK+channelsinause-dependentmanner, JPharmacolExpTher. PMID: 15947038
Titrationoftertiapin-QinhibitionofROMK1channelsbyextracellularprotons
Tertiapin-Q (TPN(Q)),ahoneybeetoxinderivative,inhibitsinward-rectifierK(+) channels bybindingtotheirexternalvestibule.InthepresentstudywefoundthatTPN(Q) inhibition ofthe channels isprofoundlyaffectedby extracellular pH.ThispHdependencemainlyreflects titration ofhistidineresidue12inTPN(Q)by extracellular protons,sinceitlargelyvanisheswhenthehistidineresidueisreplacedwithalanine.Notsurprisingly,thisalaninederivativeofTPN(Q)bindstothechannelwithmuchloweraffinity.QuantitativeThermodynamiccycleanalysisshowsthatdeprotonationofthehistidineresiduereducestheTPN(Q)-ROMK1 bindingenergyby1.6kcal/mol.ToeliminatepHsensitivitybutretainhighaffinity,wederivatizedTPN(Q)byreplacinghistidine12withlysine.Thisderivative-denotedtertiapin-KQ(TPN(KQ))-notonlyispracticallyinsensitiveto extracellular pHbutalsobindstothechannelwithevenhigheraffinitythanTPN(Q)at extracellular pH7.6.
Ramu,Y., etal.(2001)Titrationoftertiapin-QinhibitionofROMK1channelsbyextracellularprotons, Biochemistry. PMID: 11297426
TertiapinpotentlyandselectivelyblocksmuscarinicK(+)channelsinrabbitcardiacmyocytes
Tertiapin isa21-residuepeptideisolatedfromhoneybeevenoms.Arecentstudyindicatedthat tertiapin isapotentblockerofcertaintypesofinwardlyrectifying K(+)(Kir) channels ().Weexaminedtheeffectof tertiapin onionchannelcurrentsin rabbit cardiac myocytes usingthepatch-clamptechnique.Inthewhole-cellconfiguration, tertiapin fullyinhibitedacetylcholine(1microM)-induced muscarinic K(+)(K(ACh))channelcurrentsinatrialmyocytes withthehalf-maximuminhibitoryconcentrationofapproximately8nMthroughapproximately1:1stoichiometry.Thepotencyof tertiapin ininhibiting K(ACh) channels wasnotsignificantlydifferentat-40&-100mV. Tertiapin alsoinhibitedthe K(ACh)channelpreactivatedbyintracellularguanosine5′-O-(3-thiotriphosphate),anonhydrolyzableGTPanalog.AconstitutivelyactiveKirchannel,theI(K1)channel,wasatleast100timeslesssensitiveto tertiapin.AnotherKirchannelin cardiac myocytes,theATP-sensitive K(+)channel,wasvirtuallyinsensitiveto tertiapin (1microM).Thevoltage-dependent K(+)&theL-typeCa(2+) channels werenotaffectedby tertiapin (1microM).Atthesingle-channellevel, tertiapin inhibitedtheK(ACh)channelfromtheoutsideofthemembranebyreducingtheNP(o)(Nisthenumberoffunctional channels,&theP(o)istheopenprobABIlityofeachchannel)withoutaffectingthesingle-channelconductanceorfastkinetics.Therefore, tertiapin potently&selectively blocks the K(ACh)channelin cardiac myocytes inareceptor-&voltage-independentmanner. Tertiapin isanovelpharmacologicaltooltoidentifythefunctionalroleofthe K(ACh)channelintheparasympatheticregulationoftheheartbeat.
Kitamura,H., etal.(2000)TertiapinpotentlyandselectivelyblocksmuscarinicK(+)channelsinrabbitcardiacmyocytes, JPharmacolExpTher. PMID: 10734170
Mechanismsofinward-rectifierK+channelinhibitionbytertiapin-Q,Biochemistry
Tertiapin-Q(TPN(Q))isaderivativeofhoneybeetoxintertiapin(TPN)whosemethionineresidueisreplacedwithaglutamineresidue.TPN(Q)inhibitstheROMK1&GIRK1/4inward-rectifierK(+)channelswithaffinitiesverysimilartoTPN.However,unlikenativeTPN,TPN(Q)isnonoxidizablebyair.ThestabilityofTPN(Q)allowsustoinvestigatehowitinteractswiththetargetedchannels.WefoundthattheinteractionbetweenTPN(Q)&theROMK1channelisabimolecularreaction,i.e.,oneTPN(Q)moleculebindstoonechannel.TheinteractionsurfaceinTPN(Q)isprimarilyformedbyitsalphahelixratherthanthebetasheetswithwhichscorpiontoxinsformtheirinteractionsurface.Themutagenesisstudiesonboththechannel&TPN(Q)togetherstronglysuggestthattoblocktheK(+)poreTPN(Q)plugsitsalphahelixintothevestibuleoftheK(+)pore,whileleavingtheextendedstructuralportionstickingoutofthevestibuleintotheextracellularmedia.
Jin,W., etal.(1999)Mechanismsofinward-rectifierK+channelinhibitionbytertiapin-Q, Biochemistry. PMID: 10572004
Synthesisofastableformoftertiapin:ahigh-affinityinhibitorforinward-rectifierK+channels
Tertiapin (TPN),asmallproteinderivedfromhoneybeevenom,inhibitstheGIRK1/4&ROMK1 channels withnanomolaraffinities.Methionineresidue13inTPNinteractswithresidueF148inthechannel,locatedjustoutsideofthenarrowregionoftheROMK1pore.ThemethionineresidueinTPNcanbeoxidizedbyair,whichsignificantlyhindersTPNbindingtothe channels.ToovercomethereductioninTPNaffinityduetooxidationofM13,wereplacedM13inTPNwithfourteendifferentresidues.Outofthefourteenderivatives,onlytheoneinwhichM13wasreplacedbyglutamine,TPNQ,bindstothechannelwithaKivalueverysimilartothatofnativeTPN.SinceTPNQis stable&functionallyresemblesnativeTPN,itwillbeaveryusefulmolecularprobeforstudyingthe inward-rectifier K+ channels.
Jin,W.,andLu,Z.(1999)Synthesisofastableformoftertiapin:ahigh-affinityinhibitorforinward-rectifierK+channels, Biochemistry. PMID: 10572003
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