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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pH(1)=pKa+lg[c(CH₃COONa)/c(CH₃COOH)]=pKa=4.74
通HCl后,溶液是c(CH₃COOH)=0.2mol/L、c(NaCl)=0.1mol/L的混合溶液,溶液pH按照弱酸溶液pH的求法求.
c(H⁺)=√[Ka*c(CH₃COOH)]=√(10^-4.74*0.2)=0.00191(mol/L)（采用了近似公式）
pH(2)=-lg{c(H⁺)}=2.72
两个pH求得,那么pH的变化量也就可得了.pH的变化量=|pH(2)-pH(1)|=|2.72-4.74|=2.02
1）PH缓冲溶液作用原理和pH值
当往某些溶液中加入一定量的酸和碱时,有阻碍溶液pH变化的作用,称为缓冲作用,这样的溶液叫做缓冲溶液.弱酸及其盐的混合溶液（如HAc与NaAc）,弱碱及其盐的混合溶液（如NH3·H2O与NH4Cl）等都是缓冲溶液.
由弱酸HA及其盐NaA所组成的缓冲溶液对酸的缓冲作用,是由于溶液中存在足够量的碱A-的缘故.当向这种溶液中加入一定量的强酸时,H离子基本上被A-离子消耗：
所以溶液的pH值几乎不变；当加入一定量强碱时,溶液中存在的弱酸HA消耗OH-离子而阻碍pH的变化.
2）PH缓冲溶液的缓冲能力
在缓冲溶液中加入少量强酸或强碱,其溶液pH值变化不大,但若加入酸,碱的量多时,缓冲溶液就失去了它的缓冲作用.这说明它的缓冲能力是有一定限度的.
缓冲溶液的缓冲能力与组成缓冲溶液的组分浓度有关.0.1mol·L-1HAc和0.1mol·L-1NaAc组成的缓冲溶液,比0.01mol·L-1HAc和0.01mol·L-1NaAc的缓冲溶液缓冲能力大.关于这一点通过计算便可证实.但缓冲溶液组分的浓度不能太大,否则,不能忽视离子间的作用.
组成缓冲溶液的两组分的比值不为1∶1时,缓冲作用减小,缓冲能力降低,当c（盐）/c（酸）为1∶1时△pH最小,缓冲能力大.不论对于酸或碱都有较大的缓冲作用.缓冲溶液的pH值可用下式计算：
此时缓冲能力大.缓冲组分的比值离1∶1愈远,缓冲能力愈小,甚至不能起缓冲作用.对于任何缓冲体系,存在有效缓冲范围,这个范围大致在pKaφ（或pKbφ）两侧各一个pH单位之内.
弱酸及其盐（弱酸及其共轭碱）体系pH=pKaφ±1
弱碱及其盐（弱碱及其共轭酸）体系pOH=pKbφ±1
例如HAc的pKaφ为4.76,所以用HAc和NaAc适宜于配制pH为3.76～5.76的缓冲溶液,在这个范围内有较大的缓冲作用.配制pH=4.76的缓冲溶液时缓冲能力最大,此时（c（HAc）/c（NaAc）=1.
3）PH缓冲溶液的配制和应用
为了配制一定pH的缓冲溶液,首先选定一个弱酸,它的pKaφ尽可能接近所需配制的缓冲溶液的pH值,然后计算酸与碱的浓度比,根据此浓度比便可配制所需缓冲溶液.
以上主要以弱酸及其盐组成的缓冲溶液为例说明它的作用原理、pH计算和配制方法.对于弱碱及其盐组成的缓冲溶液可采用相同的方法.
PH缓冲溶液在物质分离和成分分析等方面应用广泛,如鉴定Mg2离子时,可用下面的反应：
白色磷酸铵镁沉淀溶于酸,故反应需在碱性溶液中进行,但碱性太强,可能生成白色Mg（OH）2沉淀,所以反应的pH值需控制在一定范围内,因此利用NH3·H2O和NH4Cl组成的缓冲溶液,保持溶液的pH值条件下,进行上述反应.
这就是说不用酸碱预处理吗？
Whatman的网站上没有DE52最大耐受压力，请问又经验的战友应该是多少？
Whatman的网站上：
DE32DryMicrogranularDEAECellulose
SimilarperformancecharacteristicsafterprecyclingasDE52.
DE52PreswollenMicrogranularDEAECellulose
ProbablythemostwidelyusedDEAEcelluloseintheworld;usedforbiopolymerswithlowtohighnegativecharges;exhibitsexcellentresolutionwithgoodflowrates.
附件是一本图书(MethodsinMolecularMedicine,)的章节,上面说:
WhatmanDEAE52comesalreadypreswollenandonlyneedstobetransferred
totherunningbuffer50mMTE8.
lAntibodiesUsingIonExchangeChromatography.pdf(87.06k)
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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