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
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常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
有了源数据之后把源数据按照大小排列，
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
:)
我在做一细菌不同酸碱度生长状况时,发现这些奇怪现象:pH=3的培养基灭菌(TSB液体培养基)灭菌后pH上升到到9.2!而原来pH=9.0的降到8.7(基本没多少变化),请问各位大侠,这是什么原因?
一般做不同酸碱度生长实验时,该如何才能防止pH在湿热灭菌后基本不变化?
是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
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