µ-conotoxinGIIIBisa22aminoacidconopeptideoriginallyisolatedfromthevenomofthepiscivorousmarinesnailConusgeographus.µ-conotoxinGIIIBadoptsacompactstructureconsistingofadistorted310-helixandasmallß-hairpin.µ-conotoxinGIIIBisstABIlizedbythreedisulphidebridgesandishighlyenrichedinlysineandarginineresidues,formingpotentialsitesofinteractionwithNachannels.Anunusualfeatureisthepresenceofthreehydroxyprolineresidues.µ-conotoxinGIIIBisausefulprobetodiscriminatebetweenneuronalandmusclesodiumchannelsasitexhibitsatleasta1000-foldspecificityformuscleversusnervesodiumchannels.µ-ConotoxinGIIIBselectivelyblocksNav1.4voltage-dependentsodiumchannels,whicharepredominantlyexpressedinmuscle,withanaffinitycloseto20nM.µ-ConotoxinGIIIBappearstophysicallyoccludethechannelporebybindingonsiteIoftheNa+channel.
Description:
AAsequence:Arg-Asp-Cys3-Cys4-Thr-Hyp-Hyp-Arg-Lys-Cys10-Lys-Asp-Arg-Arg-Cys15-Lys-Hyp-Met-Lys-Cys20-Cys21-Ala-NH2
Disulfidebonds:Cys3-Cys15,Cys4-Cys20andCys10-Cys21
Length(aa):22
Formula:C101H175N39O30S7
MolecularWeight:2640.26Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:
Source:Synthetic
Purityrate:>97%
Reference:
MolecularBasisofIsoform-specificμ-ConotoxinBlockofCardiac,SkeletalMuscle,andBrainNa+Channels
mu-Conotoxins(mu-CTXs)blockskeletalmuscleNa(+)channelswithanaffinity1-2ordersofmagnitudehigherthancardiacandbrainNa(+)channels.Althoughanumberofconservedporeresiduesarerecognizedascriticaldeterminantsofmu-CTXblock,themolecularbasisofisoform-specifictoxinsensitivityremainsunresolved.SequencecomparisonofthedomainII(DII)S5-S6loopsofratskeletalmuscle(mu1,Na(v)1.4),humanheart(hh1,Na(v)1.5),andratbrain(rb1,Na(v)1.1)Na(+)channelsrevealssubstantialdivergenceintheirN-terminalS5-PlinkerseventhoughtheP-S6andC-terminalPsegmentsarealmostidentical.WeusedNa(v)1.4asthebackboneandsystematicallyconvertedtheseDIIS5-PisoformvariantstothecorrespondingresiduesinNa(v)1.1andNa(v)1.5.TheNa(v)1.4–>Na(v)1.5variantsubstitutionsV724R,C725S,A728S,D730S,andC731S(Na(v)1.4numbering)reducedblockofNa(v)1.4by4-,86-,12-,185-,and55-foldrespectively,renderingtheskeletalmuscleisoformmore“cardiac-like.”Conversely,anNa(v)1.5–>Na(v)1.4chimericconstructinwhichtheNa(v)1.4DIIS5-PlinkerreplacestheanalogoussegmentinNa(v)1.5showedenhancedmu-CTXblock.However,thesevariantdeterminantsareconservedbetweenNa(v)1.1andNa(v)1.4andthuscannotexplaintheirdifferentsensitivitiestomu-CTX.ComparisonoftheirsequencesrevealstwovariantsatNa(v)1.4positions729and732:SerandAsninNa(v)1.4comparedwithThrandLysinNa(v)1.1,respectively.ThedoublemutationS729T/N732KrenderedNa(v)1.4more“brain-like”(30-folddownwardarrowinblock),andtheconversemutationT925S/K928NinNa(v)1.1reproducedthehighaffinityblockingphenotypeofNa(v)1.4.WeconcludethattheDIIS5-Plinker,althoughlyingoutsidetheconventionalion-conductingpore,playsaprominentroleinmu-CTXbinding,thusshapingisoform-specifictoxinsensitivity.
RonaldA.Li, etal.(2003)MolecularBasisofIsoform-specificμ-ConotoxinBlockofCardiac,SkeletalMuscle,andBrainNa+ Channels. JBC. PMID: 12471026
Conusgeographustoxinsthatdiscriminatebetweenneuronalandmusclesodiumchannels
Wedescribethepropertiesofafamilyof22-aminoacidpeptides,themu-conotoxins,whichareusefulprobesforinvestigatingvoltage-dependentsodiumchannelsofexcitabletissues.Themu-conotoxinsarepresentinthevenomofthepiscivorousmarinesnail,ConusgeographusL.Wehavepurifiedsevenhomologsofthemu-conotoxinsetanddeterminedtheiraminoacidsequences,asfollows,whereHyp=trans-4-hydroxyproline.GIIIAR.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2[Pro6]GIIIAR.D.C.C.T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2[Pro7]GIIIAR.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2GIIIBR.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2[Pro6]GIIIBR.D.C.C.T.P.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2[Pro7]GIIIBR.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2GIIICR.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2.Usingthemajorpeptide(GIIIA)inelectrophysiologicalstudiesonnerve-musclepreparationsandinsinglechannelstudiesusingplanarlipidbilayers,wehaveestablishedthatthetoxinblocksmusclesodiumchannels,whilehavingnodiscernIBLeeffectonnerveorbrainsodiumchannels.InbilayerstheblockingkineticsofGIIIAwerederivedbystatisticalanalysisofdiscretetransitionsbetweenblockedandunblockedstatesofbatrachotoxin-activatedsodiumchannelsfromratmuscle.Thekineticsconformtoasingle-site,reversiblebindingequilibriumwithavoltage-dependentbindingconstant.ThemeasuredvalueoftheequilibriumKDforGIIIAis100nMatOmV,decreasinge-fold/34mVofhyperpolarization.Thisvoltagedependenceofblockingissimilartothatoftetrodotoxinandsaxitoxinasmeasuredbythesametechnique.Thetissuespecificityandkineticcharacteristicssuggestthatthemu-conotoxinsmayserveasusefulligandstodistinguishsodiumchannelsubtypesindifferenttissues.
CruzLJ, etal. (1985)Conusgeographustoxinsthatdiscriminatebetweenneuronalandmusclesodiumchannels. JBC. PMID: 2410412
NovelStructuralDeterminantsofm-Conotoxin(GIIIB)BlockinRatSkeletalMuscle(m1)Na+Channels
mu-Conotoxin(mu-CTX)specificallyoccludestheporeofvoltage-dependentNa(+)channels.IntheratskeletalmuscleNa(+)channel(mu1),weexaminedthecontributionofchargedresiduesbetweenthePloopsandS6inallfourdomainstomu-CTXblock.ConversionofthenegativelychargeddomainII(DII)residuesAsp-762andGlu-765tocysteineincreasedtheIC(50)formu-CTXblockbyapproximately100-fold(wild-type=22.3+/-7.0nm;D762C=2558+/-250nm;E765C=2020+/-379nm).Restorationorreversalofchargebyexternalmodificationofthecysteine-substitutedchannelswithmethanethiosulfonatereagents(methanethiosulfonateethylsulfonate(MTSES)andmethanethiosulfonateethylammonium(MTSEA))didnotaffectmu-CTXblock(D762C:IC(50,MTSEA+)=2165.1+/-250nm;IC(50,MTSES-)=2753.5+/-456.9nm;E765C:IC(50,MTSEA+)=2200.1+/-550.3nm;IC(50,MTSES-)=3248.1+/-2011.9nm)comparedwiththeirunmodifiedcounterparts.Incontrast,thecharge-conservingmutationsD762E(IC(50)=21.9+/-4.3nm)andE765D(IC(50)=22.0+/-7.0nm)preservedwild-typeblockingbehavior,whereasthechargereversalmutantsD762K(IC(50)=4139.9+/-687.9nm)andE765K(IC(50)=4202.7+/-1088.0nm)destabilizedmu-CTXblockevenfurther,suggestingaprominentelectrostaticcomponentoftheinteractionsbetweentheseDIIresiduesandmu-CTX.Kineticanalysisofmu-CTXblockrevealsthatthechangesintoxinsensitivityarelargelyduetoacceleratedtoxindissociation(k(off))rateswithlittlechangesinassociation(k(on))rates.Weconcludethattheacidicresiduesatpositions762and765arekeydeterminantsofmu-CTXblock,primarilybyvirtueoftheirnegativecharge.TheinabilityofthebulkyMTSESorMTSEAsidechaintomodifymu-CTXsensitivityplacesstericconstraintsonthesitesoftoxininteraction.
RonaldA.Li, etal. (2000)NovelStructuralDeterminantsofm-Conotoxin(GIIIB)BlockinRatSkeletalMuscle(m1)Na+ Channels.JBC.PMID: 10859326
HyperpolarizedshiftsinthevoltagedependenceoffastinactivationofNav1.4andNav1.5inaratmodelofcriticalillnessmyopathy
Criticalillnessmyopathyisadisorderinwhichskeletalmusclebecomeselectricallyinexcitable.Wepreviouslydemonstratedthatashiftinthevoltagedependenceoffastinactivationofsodiumcurrentscontributestoinexcitabilityofaffectedfibresinananimalmodelofcriticalillnessmyopathyinwhichdenervatedratskeletalmuscleistreatedwithcorticosteroids(steroid-denervated;SD).InthecurrentstudyweexaminedwhetherexpressionofNav1.5contributestothealteredvoltagedependenceofsodiumchannelinactivationinSDmuscle.WeusedTTXandmu-conotoxinGIIIBtoselectivelyblockNav1.4inSDmuscleandfoundthatthelevelofNav1.5didnotcorrelatecloselywiththeshiftinfastinactivation.Surprisingly,wefoundthatthevoltagedependenceofinactivationofNav1.4wassimilartothatofNav1.5inskeletalmuscleinvivo.Inseverelyaffectedfibres,inactivationofbothNav1.4andNav1.5wasshiftedtowardshyperpolarizedpotentials.Weexaminedtheroleofdenervationandsteroidtreatmentintheshiftofthevoltagedependenceofinactivationandfoundthatbothdenervationandsteroidtreatmentcontributetotheshiftininactivation.OurresultssuggestthatmodulationofthevoltagedependenceofinactivationofbothNav1.4andNav1.5invivocontributestolossofelectricalexcitabilityinSDmuscle.
GregoryN.FilatovandMarkM.Rich(2004)HyperpolarizedshiftsinthevoltagedependenceoffastinactivationofNav1.4andNav1.5inaratmodelofcriticalillnessmyopathy. J.Physiol. PMID: 15254148
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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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