ω-agatoxin-IVA (ω-AGAIVA) isapeptideoriginallyisolatedfromfunnelweb-spidervenomAgelenopsisaperta.Thispeptideisaspecificblockerof P/Q-typecalciumchannel(Cav2.1). Ithasbeenreportedthat ω-agatoxinIVA isapotentblockerofvoltage-gatedcalciumchannelsininsectandvertebratecentralneurons.Thebindingsitefor ω-agatoxinIVA hasbeenlocalizedinparttotheextracellularS3–S4loopinrepeatIVoftheα-1A Ca2+ channels,whichisproximaltotheS4sensordomain.Thisiscoherentwithitsfunctionaleffect(nopore-blockingactivity,butgatingmodifierbyashiftofchannelactivationtowardsmoredepolarizedpotentials).Thismakesthistoxinavoltage-dependentblockerofP/Qcalciumchannels.
Newproductcitation
Description:
AAsequence:Lys-Lys-Lys-Cys4-Ile-Ala-Lys-Asp-Tyr-Gly-Arg-Cys12-Lys-Trp-Gly-Gly-Thr-Pro-Cys19-Cys20-Arg-Gly-Arg-Gly-Cys25-Ile-Cys27-Ser-Ile-Met-Gly-Thr-Asn-Cys34-Glu-Cys36-Lys-Pro-Arg-Leu-Ile-Met-Glu-Gly-Leu-Gly-Leu-Ala-OH
Disulfidebonds:Cys4-Cys20,Cys12-Cys25,Cys19-Cys36andCys27-Cys34
Length(aa):48
Formula:C217H360N68O60S10
MolecularWeight:5202.48Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:145017-83-0
Source:Synthetic
Purityrate:>97%
Reference:
P-typecalciumchannelsblockedbythespidertoxinomega-Aga-IVA
Voltage-dependentcalciumchannelsmediatecalciumentryintoneurons,whichiscrucialformanyprocessesinthebrainincludingsynaptictransmission,dendriticspiking,geneexpressionandcelldeath.Manytypesofcalciumchannelsexistinmammalianbrains,buthigh-affinityblockersareavailableforonlytwotypes,L-typechannels(targetedbynimodipineandotherdihydropyridinechannelblockers)andN-typechannels(targetedbyomega-conotoxin).Inasearchfornewchannelblockers,wehaveidentifiedapeptidetoxinfromfunnelwebspidervenom,omega-Aga-IVA,whichisapotentinhibitorofbothcalciumentryintoratbrainsynaptosomesandof‘P-type’calciumchannelsinratPurkinjeneurons.omega-Aga-IVAwillfacilitatecharacterizationofbraincalciumchannelsresistanttoexistingchannelblockersandmayassistinthedesignofneuroprotectivedrugs.
MintzI.M., etal. (1992)P-typecalciumchannelsblockedbythespidertoxinomega-Aga-IVA. Nature . PMID:1311418
Splicingofalpha1AsubunitgenegeneratesphenotypicvariantsofP-andQ-typecalciumchannels
P-typeandQ-typecalciumchannelsmediateneurotransmitterreleaseatmanysynapsesinthemammaliannervoussystem.Thealpha1Acalciumchannelhasbeenimplicatedintheetiologiesofconditionssuchasepisodicataxia,epilepsyandfamilialmigraine,andsharesseveralpropertieswithnativeP-andQ-typechannels.However,theexactrelationshipbetweenalpha1AandP-andQ-typechannelsisunknown.Herewereportthatalternativesplicingofthealpha1Asubunitgeneresultsinchannelswithdistinctkinetic,pharmacologicalandmodulatoryproperties.Overall,theresultsindicatethatalternativesplicingofthealpha1AgenegeneratesP-typeandQ-typechannelsaswellasmultiplephenotypicvariants.
BourinetE,etal.(1999)Splicingofalpha1AsubunitgenegeneratesphenotypicvariantsofP-andQ-typecalciumchannels. NatNeurosci. PMID:10321243
Agatoxins:ionchannelspecifictoxinsfromtheAmericanfunnelwebspider,Agelenopsisaperta
AgatoxinsfromAgelenopsisapertavenomtargetthreeclassesofionchannels,includingtransmitter-activatedcationchannels,voltage-activatedsodiumchannels,andvoltage-activatedcalciumchannels.Thealpha-agatoxinsarenon-competitive,use-dependentantagoNISTsofglutamatereceptorchannels,andproducerapidbutreversIBLeparalysisininsectprey.Theiractionsarefacilitatedbythemicro-agatoxins,whichshiftvoltage-dependentactivationofneuronalsodiumchannelstomorenegativepotentials,causingspontaneoustransmitterreleaseandrepetitiveactionpotentials.Theomega-agatoxinstargetneuronalcalciumchannels,modifyingtheirpropertiesindistinctways,eitherthroughgatingmodification(omega-Aga-IVA)orbyreductionofunitarycurrent(omega-Aga-IIIA).Thealpha-agatoxinsandomega-agatoxinsmodifybothinsectandvertebrateionchannels,whilethemicro-agatoxinsareselectiveforinsectchannels.Agatoxinshavebeenusedasselectivepharmacologicalprobesforcharacterizationofionchannelsinthebrainandheart,andhavebeenevaluatedascandidatebiopesticides.
AdamsME.(2004)Agatoxins:ionchannelspecifictoxinsfromtheAmericanfunnelwebspider,Agelenopsisaperta.Toxicon. PMID:15066410
Molecularpharmacologyofhighvoltage-activatedcalciumchannels
Voltage-gatedcalciumchannelsarekeysourcesofcalciumentryintothecytosolofmanyexcitabletissues.Anumberofdifferenttypesofcalciumchannelshavebeenidentifiedandshowntomediatespecializedcellularfunctions.Becauseoftheirfundamentalnature,theyareimportanttargetsfortherapeuticinterventionindisorderssuchashypertension,pain,stroke,andepilepsy.Calciumchannelantagonistsfallintooneofthefollowingthreegroups:smallinorganicions,largepeptideblockers,andsmallorganicmolecules.Inorganicionsnonselectivelyinhibitcalciumentrybyphysicalporeocclusionandareoflittletherapeuticvalue.Calcium-channel-blockingpeptidesisolatedfromvariouspredatoryanimalssuchasspidersandconesnailsareoftenhighlyselectiveblockersofindividualtypesofcalciumchannels,eitherbypreventingcalciumfluxthroughtheporeorbyantagonizingchannelactivation.Therearemanystructure-activity-relationclassesofsmallorganicmoleculesthatinteractwithvarioussitesonthecalciumchannelprotein,withactionsrangingfromselectivehighaffinityblocktorelativelynondiscriminatoryactiononmultiplecalciumchannelisoforms.Detailedinteractionswiththecalciumchannelproteinarewellunderstoodforthedihydropyridineandphenylalkylaminedrugclasses,whereasweareonlybeginningtounderstandthemolecularactionsofsomeofthemorerecentlydiscoveredcalciumchannelblockers.Here,weprovideacomprehensivereviewofpharmacologyofhighvoltage-activatedcalciumchannels.
DoeringCJ,ZamponiGW.(2003)Molecularpharmacologyofhighvoltage-activatedcalciumchannels. JBioenergBiomembr. PMID:15000518
Ahotspotfortheinteractionofgatingmodifiertoxinswithvoltage-dependentionchannels
Thegatingmodifiertoxinsarealargefamilyofproteintoxinsthatmodifyeitheractivationorinactivationofvoltage-gatedionchannels.omega-Aga-IVAisagatingmodifiertoxinfromspidervenomthatinhibitsvoltage-gatedCa(2+)channelsbyshiftingactivationtomoredepolarizedvoltages.WeidentifiedtwoGluresiduesneartheCOOH-terminaledgeofS3inthealpha(1A)Ca(2+)channel(oneinrepeatIandtheotherinrepeatIV)thatalignwithGluresiduespreviouslyimplicatedinformingthebindingsitesforgatingmodifiertoxinsonK(+)andNa(+)channels.WefoundthatmutationoftheGluresidueinrepeatIoftheCa(2+)channelhadnosignificanteffectoninhibitionbyomega-Aga-IVA,whereastheequivalentmutationoftheGluinrepeatIVdisruptedinhibitionbythetoxin.TheseresultssuggestthattheCOOH-terminalendofS3withinrepeatIVcontributestoformingareceptorforomega-Aga-IVA.ThestrongpredictivevalueofpreviousmappingstudiesforK(+)andNa(+)channeltoxinsarguesforaconservedbindingmotifforgatingmodifiertoxinswithinthevoltage-sensingdomainsofvoltage-gatedionchannels.
WinterfieldJR,SwartzKJ.(2000)Ahotspotfortheinteractionofgatingmodifiertoxinswithvoltage-dependentionchannels. JGenPhysiol. PMID:11055992
ModulationofinsectCa(v)channelsbypeptidicspidertoxins
Insectshaveamuchsmallerrepertoireofvoltage-gatedcalcium(Ca(V))channelsthanvertebrates.DrosophilamelanogasterharborsonlyasingleorthologofeachofthevertebrateCa(V)1,Ca(V)2,andCa(V)3subtypes,althoughitsbasalinventoryisexpandedbyalternativesplicingandeditingofCa(V)channeltranscripts.Nevertheless,thereappearstobelittlefunctionalplasticitywithinthislimitedpanelofinsectCa(V)channels,sincesevereloss-of-functionmutationsingenesencodingthepore-formingalpha1subunitsinDrosophilaareembryoniclethal.Sincetheprimaryroleofspidervenomistoparalyzeorkillinsectprey,itisnotsurprisingthatmost,ifnotall,spidervenomscontainpeptidesthatpotentlymodifytheactivityofthesefunctionallycriticalinsectCa(V)channels.Unfortunately,ithasprovendifficulttodeterminethepreciseionchannelsubtypesrecognizedbythesepeptidetoxinssinceinsectCa(V)channelshavesignificantlydifferentpharmacologytotheirvertebratecounterparts,andclonedinsectCa(V)channelsarenotavailableforelectrophysiologicalstudies.However,biochemicalandgeneticstudiesindicatethatsomeofthesespidertoxinsmightultimatelybecomethedefiningpharmacologyforcertainsubtypesofinsectCa(V)channels.ThisreviewfocusesonpeptidicspidertoxinsthatspecificallytargetinsectCa(V)channels.Inadditiontoprovidingnovelmoleculartoolsforionchannelcharacterization,someofthesetoxinsarebeingusedasleadstodevelopnewmethodsforcontrollinginsectpests.
KingGF.(2007)ModulationofinsectCa(v)channelsbypeptidicspidertoxins. Toxicon. PMID:17197008
omega-ToxinsaffectNa+currentsinneurosecretoryinsectneurons
omega-toxinsspecificallyblockcertainCa2+channelsinmammalianneuronsaswellasindorsalunpairedmedianneuronsisolatedfromthecockroachPeriplanetaamericana.InthesecockroachneuronsboththeP/Q-typeblockersomega-agatoxinIVAandomega-conotoxinMVIICbutnottheN-typeCa2+channelblockeromega-conotoxinGVIAaffectedfastNa+currentssensitivetotetrodotoxinandveratridine.Bothomega-toxinsenhancedNa+currentdecayandthusdecreasedtheamplitudesofthepeakcurrents.Theyalsoledtoaslowerrecoveryfrominactivation.Toxineffectsdevelopingwithinafewminwereotremoveduponwashing.Theywerenotuse-dependent.Thedescriptionoftheeffectofomega-conotoxinMVIIConcurrentkineticsintermsoftheHodgkin-Huxleymodelrevealedthatsteady-stateparameterswerenotaffectedwhereasthetimeconstantofinactivationwasconsiderablyreduced.Undercontrolconditions,theinactivationtimeconstantissimilartothetimeconstantofrecoveryfrominactivation.Thetoxin-inducedincreaseofthelattertimeconstantandthedecreaseoftheinactivationtimeconstantindicatethatinactivationcannolongerbedescribedbyfirst-orderkinetics.
WicherD,PenzlinH.(1998)omega-ToxinsaffectNa+currentsinneurosecretoryinsectneurons. ReceptorsChannels. PMID:9826912
Ca2+currentsincentralinsectneurons:electrophysiologicalandpharmacologicalproperties
Ca2+currentsindorsalunpairedmedian(DUM)neuronsisolatedfromthefifthaBDominalganglionofthecockroachPeriplanetaamericanawereinvestigatedwiththewholecellpatch-clamptechnique.Onthebasisofkineticandpharmacologicalproperties,twodifferentCa2+currentswereseparatedinthesecells:mid/low-voltage-activated(M-LVA)currentsandhigh-voltage-activated(HVA)currents.M-LVAcurrentshadanactivationthresholdof-50mVandreachedmaximalpeakvaluesat-10mV.Theyweresensitivetodepolarizedholdingpotentialsanddecayedveryrapidly.ThedecaywaslargelyCa2+dependent.M-LVAcurrentswereeffectivelyblockedbyCd2+medianinhibitingconcentration(IC50=9microM),buttheyalsohadaremarkablesensitivitytoNi2+(IC50=19microM).M-LVAcurrentswereinsensitivetovertebrateLVAchannelblockerslikeflunarizineandamiloride.Thecurrentswere,however,potentlyblockedbyomega-conotoxinMVIIC(1microM)andomega-agatoxinIVA(50nM).Theblockingeffectsofomega-toxinsdevelopedfast(timeconstanttau=15s)andwerefullyreversibleafterwash.HVAcurrentsactivatedpositiveto-30mVandshowedmaximalpeakcurrentsat+10mV.Theywereresistanttodepolarizedholdingpotentialsupto-50mVanddecayedinalesspronouncedmannerthanM-LVAcurrents.HVAcurrentswerepotentlyblockedbyCd2+(IC50=5microM)butlessaffectedbyNi2+(IC50=40microM).Thesecurrentswerereducedbyphenylalkylamineslikeverapamil(10microM)andbenzothiazepineslikediltiazem(10microM),buttheywereinsensitivetodihydropyridineslikenifedipine(10microM)andBAYK8644(10microM).FurThermore,HVAcurrentsweresensitivetoomega-conotoxinGVIA(1microM).Thetoxin-inducedreductionofcurrentsappearedslowly(tauapproximately120s)andtherecoveryafterwashwasincompleteinmostcases.Thedihydropyridineinsensitivityofthephenylalkylamine-sensitiveHVAcurrentsisapropertythecockroachDUMcellssharewithotherinvertebrateneurons.ComparedwithCa2+currentsinvertebrates,theDUMneuroncurrentdifferconsiderablyfromthepresentlyknowntypes.Althoughtherearesomesimilaritiesconcerningkinetics,thepharmacologicalprofileofthecockroachCa2+currentsespeciallyisverydifferentfromprofilesalreadydescribedforvertebratecurrents.
WicherD,PenzlinH.(1997)Ca2+currentsincentralinsectneurons:electrophysiologicalandpharmacologicalproperties. JNeurophysiol. PMID:9120560
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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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