Stromatoxin-1(ScTx-1) hasbeenisolatedfromthevenomoftheAfricantarentula Stromatopelmacalceata.Stromatoxin-1 isa34amino-acidlongpeptidethatbelongstothestructuralfamilyofinhibitorcystineknotpeptidesreticulatedbythreedisulfidebridges.IthasanamidatedC-terminusandbearsstronghomologywithhanatoxin1(83%).Stromatoxin-1 inhibitswithhighaffinitiesKv2.1andKv2.2,thatencodedelayedK+ channels(respectively,withIC50 of12and21nM).Theblockisvoltage-dependentandslowlyreversIBLe. Stromatoxin-1 isalsoaverysensitiveinhibitorofKv4.2,thatencodesatransientK+ current(IC50 of1.2nM).Herealso,theblockisvoltage-dependentindicatingthatScTx-1actsasagatingmodifierratherthanaporeblocker.ReversibilityisfasteronKv4.2channels.Incontrast,Stromatoxin-1 hasnoeffectonKv1.1,Kv1.2,Kv1.3,Kv1.4,Kv1.5,Kv1.6orKv3.4channels.Thetoxinhasalsonoeffectonvoltage-dependentNa+ andCa2+ channelsofcerebellargranulecells. Stromatoxin-1 wasfoundtoincreasethespontaneousphasiccontractionamplitude,muscleforceandtoneinisolatedraturinarybladdersmoothmuscle.Italsoenhancesmyogenicconstrictioninpressurizedarterialsegments.
Description:
AAsequence: Asp-Cys2-Thr-Arg-Met-Phe-Gly-Ala-Cys9-Arg-Arg-Asp-Ser-Asp-Cys15-Cys16-Pro-His-Leu-Gly-Cys21-Lys-Pro-Thr-Ser-Lys-Tyr-Cys28-Ala-Trp-Asp-Gly-Thr-Ile-NH2
Disulfidebonds:Cys2-Cys16,Cys9-Cys21 andCys15-Cys28
Length(aa):34
Formula:C156H237N49O48S7
MolecularWeight: 3791,3Da
Appearance:whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:
Source:Synthetic
Purityrate:>98%
Reference:
Kv2dysfunctionafterperipheralaxotomyenhancessensoryneuronresponsivenesstosustainedinput
PeripheralnerveinjuriescausedbytraumaareassociatedwithincreasedsensoryneuronexcitABIlityanddebilitatingchronicpainsymptoms.Axotomy-inducedalterationsinthefunctionofionchannelsarethoughttolargelyunderliethepathophysiologyofthesephenotypes.Here,wecharacterisethemRNAdistributionofKv2familymembersinratdorsalrootganglia(DRG)anddescribealinkbetweenKv2functionandmodulationofsensoryneuronexcitability.Kv2.1andKv2.2wereamplyexpressedincellsofallsizes,beingparticularlyabundantinmedium-largeneuronsalsoimmunoreactiveforneurofilament-200.Peripheralaxotomyledtoarapid,robustandlong-lastingtranscriptionalKv2downregulationintheDRG,correlatedwiththeonsetofmechanicalandthermalhypersensitivity.TheconsequencesofKv2loss-of-functionweresubsequentlyinvestigatedinmyelinatedneuronsusingintracellularrecordingsonexvivoDRGpreparations.Innaïveneurons,pharmacologicalKv2.1/Kv2.2inhibitionbystromatoxin-1(ScTx)resultedinshorteningofactionpotential(AP)after-hyperpolarization(AHP).Incontrast,ScTxapplicationonaxotomizedneuronsdidnotalterAHPduration,consistentwiththeinjury-inducedKv2downregulation.InaccordancewithashortenedAHP,ScTxtreatmentalsoreducedtherefractoryperiodandimprovedAPconductiontothecellsomaduringhighfrequencystimulation.TheseresultssuggestthatKv2downregulationfollowingtraumaticnervelesionfacilitatesgreaterfidelityofrepetitivefiringduringprolongedinputandthusnormalKv2functionispostulatedtolimitneuronalexcitability.Insummary,wehaveprofiledKv2expressioninsensoryneuronsandprovideevidenceforthecontributionofKv2dysfunctioninthegenerationofhyperexcitablephenotypesencounteredinchronicpainstates.
TsantoulasC., etal. (2014) Kv2dysfunctionafterperipheralaxotomyenhancessensoryneuronresponsivenesstosustainedinput. ExpNeurol. PMID:24252178
Voltage-gatedK+channelssensitivetostromatoxin-1regulatemyogenicandneurogeniccontractionsofraturinarybladdersmoothmuscle
Membersofthevoltage-gatedK(+)(K(V))channelfamilyaresuggestedtocontroltherestingmembranepotentialandtherepolarizationphaseoftheactionpotentialinurinarybladdersmoothmuscle(UBSM).Recentstudiesreportthatstromatoxin-1,apeptideisolatedfromtarantulas,selectivelyinhibitsK(V)2.1,K(V)2.2,K(V)4.2,andK(V)2.1/9.3channels.TheobjectiveofthisstudywastoinvestigatewhetherK(V)channelssensitivetostromatoxin-1participateintheregulationofratUBSMcontractilityandtoidentifytheirmolecularfingerprints.Stromatoxin-1(100nM)increasedthespontaneousphasiccontractionamplitude,muscleforce,andtoneinisolatedUBSMstrips.However,stromatoxin-1(100nM)hadnoeffectontheUBSMcontractionsinducedbydepolarizingagentssuchasKCl(20mM)orcarbachol(1microM).Thisindicatesthat,underconditionsofsustainedmembranedepolarization,theK(V)channelssensitivetostromatoxin-1havenofurthercontributiontothemembraneexcitabilityandcontractility.Stromatoxin-1(100nM)increasedtheamplitudeoftheelectricalfieldstimulation-inducedcontractions,suggestingalsoaroleforthesechannelsinneurogeniccontractions.RT-PCRexperimentsonfreshlyisolatedUBSMcellsshowedmRNAexpressionofK(V)2.1,K(V)2.2,andK(V)9.3,butnotK(V)4.2channelsubunits.ProteinexpressionofK(V)2.1andK(V)2.2channelswasdetectedusingWesternblotandwasfurtherconfirmedbyimmunocytochemicaldetectioninfreshlyisolatedUBSMcells.ThesenovelfindingsindicatethatK(V)2.1andK(V)2.2,butnotK(V)4.2,channelsubunitsareexpressedinratUBSMandplayakeyroleinopposingbothmyogenicandneurogenicUBSMcontractions.
ChenM., etal. (2013) Voltage-gatedK+ channelssensitivetostromatoxin-1regulatemyogenicandneurogeniccontractionsofraturinarybladdersmoothmuscle. AmJPhysiolRegulIntegrCompPhysiol. PMID:20393158
KV2.1andelectricallysilentKVchannelsubunitscontrolexcitabilityandcontractilityofguineapigdetrusorsmoothmuscle
Voltage-gatedK(+)(K(V))channelsareimplicatedindetrusorsmoothmuscle(DSM)function.However,littleisknownaboutthefunctionalroleoftheheterotetramericK(V)channelsinDSM.Inthisreport,weprovidemolecular,electrophysiological,andfunctionalevidenceforthepresenceofK(V)2.1andelectricallysilentK(V)channelsubunitsinguineapigDSM.Stromatoxin-1(ScTx1),aselectiveinhibitorofthehomotetramericK(V)2.1,K(V)2.2,andK(V)4.2aswellastheheterotetramericK(V)2.1/6.3andK(V)2.1/9.3channels,wasusedtoexaminetheroleoftheseK(V)channelsinDSMfunction.RT-PCRindicatedmRNAexpressionofK(V)2.1,K(V)6.2-6.3,K(V)8.2,andK(V)9.1-9.3subunitsinisolatedDSMcells.K(V)2.1proteinexpressionwasconfirmedbyWesternblotandimmunocytochemistry.Perforatedwholecellpatch-clampexperimentsrevealedthatScTx1(100nM)inhibitedtheamplitudeoftheK(V)currentinfreshlyisolatedDSMcells.ScTx1(100nM)didnotsignificantlychangethesteady-stateactivationandinactivationcurvesforK(V)current.However,ScTx1(100nM)decreasedtheactivationtime-constantoftheK(V)currentatpositivevoltages.Althoughourpatch-clampdatacouldnotexcludethepresenceofthehomotetramericK(V)2.1channels,thebiophysicalcharacteristicsoftheScTx1-sensitivecurrentwereconsistentwiththepresenceofheterotetramericK(V)2.1/silentK(V)channels.Current-clamprecordingsshowedthatScTx1(100nM)didnotchangetheDSMcellrestingmembranepotential.ScTx1(100nM)increasedthespontaneousphasiccontractionamplitude,muscleforce,andmuscletoneaswellastheamplitudeoftheelectricalfieldstimulation-inducedcontractionsofisolatedDSMstrips.Collectively,ourdatarevealedthatK(V)2.1-containingchannelsareimportantphysiologicalregulatorsofguineapigDSMexcitabilityandcontractility.
HristovKL., etal. (2012) KV2.1andelectricallysilentKVchannelsubunitscontrolexcitabilityandcontractilityofguineapigdetrusorsmoothmuscle. AmJPhysiolCellPhysiol.PMID:21998137
MechanismofestrADIol-inducedblockofvoltage-gatedK+currentsinratmedialpreopticneurons
Thepresentstudywasconductedtocharacterizepossiblerapideffectsof17-β-estradiolonvoltage-gatedK(+)channelsinpreopticneuronsand,inparticular,toidentifythemechanismsbywhich17-β-estradiolaffectstheK(+)channels.Whole-cellcurrentsfromdissociatedratpreopticneuronswerestudiedbyperforated-patchrecording.17-β-Estradiolrapidly(withinseconds)andreversiblyreducedtheK(+)currents,showinganEC(50)valueof9.7µM.Theeffectwasslightlyvoltagedependent,butindependentofexternalCa(2+),andnotsensitivetoanestrogen-receptorblocker.Although17-α-estradiolalsosignificantlyreducedtheK(+)currents,membrane-impermeantformsofestradioldidnotreducetheK(+)currentsandotherestrogens,testosteroneandcholesterolwereconsiderablylesseffective.ThereductioninducedbyestradiolwasoverlappingwiththatoftheK(V)-2-channelblockerr-stromatoxin-1.ThetimecourseofK(+)currentin17-β-estradiol,withatime-dependentinhibitionandaslightdependenceonexternalK(+),suggestedanopen-channelblockmechanism.Thepropertiesofblockwerepredictedfromacomputationalmodelwhere17-β-estradiolbindstoopenK(+)channels.Itwasconcludedthat17-β-estradiolrapidlyreducesvoltage-gatedK(+)currentsinawayconsistentwithanopen-channelblockmechanism.Thissuggestsanewmechanismforsteroidactiononionchannels.
DruzinM., etal. (2011) Mechanismofestradiol-inducedblockofvoltage-gatedK+currentsinratmedialpreopticneurons. PLoSOne. PMID:21625454
PostnataldevelopmentofA-typeandKv1-andKv2-mediatedpotassiumchannelcurrentsinneocorticalpyramidalneurons
Potassiumchannelsregulatenumerousaspectsofneuronalexcitability,andseveralvoltage-gatedK(+)channelsubunitshavebeenidentifiedinpyramidalneuronsofratneocortex.Previousstudieshaveeitherconsideredthedevelopmentofoutwardcurrentasawholeordividedcurrentsintotransient,A-typeandpersistent,delayedrectifiercomponentsbutdidnotdifferentiatebetweencurrentcomponentsdefinedbyα-subunittype.TofacilitatecomparisonsofstudiesreportingK(+)currentsfromanimalsofdifferentagesandtounderstandthefunctionalrolesofspecificcurrentcomponents,wecharacterizedthepostnataldevelopmentofidentifiedKvchannel-mediatedcurrentsinpyramidalneuronsfromlayersII/IIIfromratsomatosensorycortex.Boththepersistent/slowlyinactivatingandtransientcomponentsofthetotalK(+)currentincreasedindensitywithpostnatalage.WeusedspecificpharmacologicalagentstotesttherelativecontributionsofputativeKv1-andKv2-mediatedcurrents(100nMα-dendrotoxinand600nMstromatoxin,respectively).Acombinationofvoltageprotocol,pharmacology,andcurvefittingwasusedtoisolatetherapidlyinactivatingA-typecurrent.Wefoundthatthedensityofallidentifiedcurrentcomponentsincreasedwithpostnatalage,approachingaplateauat3-5wk.Wefoundnosignificantchangesintherelativeproportionsorkineticsofanycomponentbetweenpostnatalweeks1and5,exceptthattheactivationtimeconstantforA-typecurrentwaslongerat1wk.TheputativeKv2-mediatedcomponentwasthelargestatallages.ImmunocytochemistryindicatedthatproteinexpressionforKv4.2,Kv4.3,Kv1.4,andKv2.1increasedbetween1wkand4-5wkofage.
GuanD., etal. (2011) PostnataldevelopmentofA-typeandKv1-andKv2-mediatedpotassiumchannelcurrentsinneocorticalpyramidalneurons. JNeurophysiol.PMID:21451062
Stromatoxin-sensitive,heteromultimericKv2.1/Kv9.3channelscontributetomyogeniccontrolofcerebralarterialdiameter
Cerebralvascularsmoothmusclecontractilityplaysacrucialroleincontrollingarterialdiameterand,thereby,bloodflowregulationinthebrain.AnumberofK(+)channelshavebeensuggestedtocontributetotheregulationofdiameterbycontrollingsmoothmusclemembranepotential(E(m))andCa(2+)influx.Previousstudiesindicatethatstromatoxin(ScTx1)-sensitive,Kv2-containingchannelscontributetothecontrolofcerebralarterialdiameterat80mmHg,buttheirpreciseroleandmolecularcompositionwerenotdetermined.Here,wetestedifKv2subunitsassociatewith‘silent’subunitsfromtheKv5,Kv6,Kv8orKv9subfamiliestoformheterotetramericchannelsthatcontributetocontrolofdiameterofratmiddlecerebralarteries(RMCAs)overarangeofintraluminalpressurefrom10to100mmHg.ThepredominantmRNAsexpressedbyRMCAsencodeKv2.1andKv9.3subunits.Co-localizationofKv2.1andKv9.3proteinsattheplasmamembraneofdissociatedsingleRMCAmyocyteswasdetectedbyproximityligationassay.ScTx1-sensitivenativecurrentofRMCAmyocytesandKv2.1/Kv9.3currentsexhibitedfunctionalidentitybasedonthesimilarityoftheirdeactivationkineticsandvoltagedependenceofactivationthatweredistinctfromthoseofhomomultimericKv2.1channels.ScTx1treatmentenhancedthemyogenicresponseofpressurizedRMCAsbetween40and100mmHg,butthistoxinalsocausedconstrictionbetween10and40mmHgthatwasnotpreviouslyobservedfollowinginhibitionoflargeconductanceCa(2+)-activatedK(+)(BK(Ca))andKv1channels.Takentogether,thisstudydefinesthemolecularbasisofKv2-containingchannelsandcontributestoourunderstandingofthefunctionalsignificanceoftheirexpressionincerebralvasculature.Specifically,ourfindingsprovidethefirstevidenceofheteromultimericKv2.1/Kv9.3channelexpressioninRMCAmyocytesandtheirdistinctcontributiontocontrolofcerebralarterialdiameteroverawiderrangeofE(m)andtransmuralpressurethanKv1orBK(Ca)channelsowingtotheirnegativerangeofvoltage-dependentactivation.
ZhongXZ., etal. (2010) Stromatoxin-sensitive,heteromultimericKv2.1/Kv9.3channelscontributetomyogeniccontrolofcerebralarterialdiameter. JPhysiol. PMID:20876197
ParticipationofKCNQ(Kv7)potassiumchannelsinmyogeniccontrolofcerebralarterialdiameter
KCNQgeneexpressionwaspreviouslyshowninvariousrodentbloodvessels,wheretheproductsofKCNQ4andKCNQ5,Kv7.4andKv7.5potassiumchannelsubunits,respectively,haveaninfluenceonvascularreactivity.TheaimofthisstudywastodetermineifsmallcerebralresistancearteriesoftheratexpressKCNQgenesandwhetherKv7channelsparticipateintheregulationofmyogeniccontrolofdiameter.Quantitativereversetranscriptionpolymerasechainreaction(QPCR)wasundertakenusingRNAisolatedfromratmiddlecerebralarteries(RMCAs)andimmunocytochemistrywasperformedusingKv7subunit-specificantibodiesandfreshlyisolatedRMCAmyocytes.KCNQ4messagewasmoreabundantthanKCNQ5=KCNQ1,butKCNQ2andKCNQ3messagelevelswerenegligible.Kv7.1,Kv7.4andKv7.5immunoreactivitywaspresentatthesarcolemmaoffreshlyisolatedRMCAmyocytes.Linopirdine(1microm)partiallydepressed,whereastheKv7activatorS-1(3and/or20microm)enhancedwhole-cellKv7.4(inHEK293cells),aswellasnativeRMCAmyocyteKvcurrentamplitude.TheeffectsofS-1werevoltage-dependent,withprogressivelossofstimulationatpotentialsof>15mV.AttheconcentrationsemployedlinopirdineandS-1didnotaltercurrentsduetorecombinantKv1.2/Kv1.5orKv2.1/Kv9.3channels(inHEK293cells)thatarealsoexpressedbyRMCAmyocytes.Incontrast,anotherwidelyusedKv7blocker,XE991(10microm),significantlyattenuatednativeKvcurrentandalsoreducedKv1.2/Kv1.5andKv2.1/Kv9.3currents.PressurizedarterialmyographywasperformedusingRMCAsexposedtointravascularpressuresof10-100mmHg.Linopirdine(1microm)enhancedthemyogenicresponseat20mmHg,whereastheactivationofKv7channelswithS-1(20microm)inhibitedmyogenicconstrictionat>20mmHgandreversedtheincreasedmyogenicresponseproducedbysuppressionofKv2-containingchannelswith30nmstromatoxin(ScTx1).ThesedatarevealanovelcontributionofKCNQgeneproductstotheregulationofmyogeniccontrolofcerebralarterialdiameterandsuggestthatKv7channelactivatingdrugsmaybeappropriatecandidatesforthedevelopmentofaneffectivetherapytoamelioratecerebralvasospasm.
ZhongXZ.,etal.(2010) ParticipationofKCNQ(Kv7)potassiumchannelsinmyogeniccontrolofcerebralarterialdiameter.JPhysiol.PMID:20624791
Membranepotential-dependentinactivationofvoltage-gatedionchannelsinalpha-cellsinhibitsglucagonsecretionfromhumanislets
OBJECTIVE:
Todocumentthepropertiesofthevoltage-gatedionchannelsinhumanpancreaticalpha-cellsandtheirroleinglucagonrelease.
RESEARCHDESIGNANDMETHODS:
Glucagonreleasewasmeasuredfromintactislets.[Ca(2+)](i)wasrecordedincellsshowingspontaneousactivityat1mmol/lglucose.Membranecurrentsandpotentialweremeasuredbywhole-cellpatch-clampinginisolatedalpha-cellsidentifiedbyimmunocytochemistry.
RESULT:
Glucoseinhibitedglucagonsecretionfromhumanislets;maximalinhibitionwasobservedat6mmol/lglucose.Glucagonsecretionat1mmol/lglucosewasinhibitedbyinsulinbutnotbyZnCl(2).GlucoseremainedinhibitoryinthepresenceofZnCl(2)andafterblockadeoftype-2somatostatinreceptors.Humanalpha-cellsareelectricallyactiveat1mmol/lglucose.InhibitionofK(ATP)-channelswithtolbutamidedepolarizedalpha-cellsby10mVandreducedtheactionpotentialamplitude.Humanalpha-cellscontainheteropodatoxin-sensitiveA-typeK(+)-channels,stromatoxin-sensitivedelayedrectifyingK(+)-channels,tetrodotoxin-sensitiveNa(+)-currents,andlow-thresholdT-type,isradipine-sensitiveL-type,andomega-agatoxin-sensitiveP/Q-typeCa(2+)-channels.Glucagonsecretionat1mmol/lglucosewasinhibitedby40-70%bytetrodotoxin,heteropodatoxin-2,stromatoxin,omega-agatoxin,andisradipine.The[Ca(2+)](i)oscillationsdependprincipallyonCa(2+)-influxviaL-typeCa(2+)-channels.Capacitancemeasurementsrevealedarapid(<50ms)componentofexocytosis.Exocytosiswasnegligibleatvoltagesbelow-20mVandpeakedat0mV.BlockingP/Q-typeCa(2+)-currentsabolisheddepolarization-evokedexocytosis.
CONCLUSIONS:
Humanalpha-cellsareelectricallyexcitable,andblockadeofanyionchannelinvolvedinactionpotentialdepolarizationorrepolarizationresultsininhibitionofglucagonsecretion.Weproposethatvoltage-dependentinactivationofthesechannelsunderliestheinhibitionofglucagonsecretionbytolbutamideandglucose.
RamracheyaR.,etal.(2010) Membranepotential-dependentinactivationofvoltage-gatedionchannelsinalpha-cellsinhibitsglucagonsecretionfromhumanislets.Diabetes.PMID:20547976
Voltage-gatedionchannelsinhumanpancreaticbeta-cells:electrophysiologicalcharacterizationandroleininsulinsecretion
OBJECTIVE:
Tocharacterizethevoltage-gatedionchannelsinhumanbeta-cellsfromnondiabeticdonorsandtheirroleinglucose-stimulatedinsulinrelease.
RESEARCHDESIGNANDMETHODS:
Insulinreleasewasmeasuredfromintactislets.Whole-cellpatch-clampexperimentsandmeasurementsofcellcapacitancewereperformedonisolatedbeta-cells.TheionchannelcomplementwasdeterminedbyquantitativePCR.
RESULTS:
Humanbeta-cellsexpresstwotypesofvoltage-gatedK(+)currentsthatflowthroughdelayedrectifying(K(V)2.1/2.2)andlarge-conductanceCa(2+)-activatedK(+)(BK)channels.BlockadeofBKchannels(usingiberiotoxin)increasedactionpotentialamplitudeandenhancedinsulinsecretionby70%,whereasinhibitionofK(V)2.1/2.2(withstromatoxin)waswithoutstimulatoryeffectonelectricalactivityandsecretion.Voltage-gatedtetrodotoxin(TTX)-sensitiveNa(+)currents(Na(V)1.6/1.7)contributetotheupstrokeofactionpotentials.InhibitionofNa(+)currentswithTTXreducedglucose-stimulated(6-20mmol/l)insulinsecretionby55-70%.Humanbeta-cellsareequippedwithL-(Ca(V)1.3),P/Q-(Ca(V)2.1),andT-(Ca(V)3.2),butnotN-orR-typeCa(2+)channels.BlockadeofL-typechannelsabolishedglucose-stimulatedinsulinrelease,whileinhibitionofT-andP/Q-typeCa(2+)channelsreducedglucose-induced(6mmol/l)secretionby60-70%.MembranepotentialrecordingssuggestthatL-andT-typeCa(2+)channelsparticipateinactionpotentialgeneration.BlockadeofP/Q-typeCa(2+)channelssuppressedexocytosis(measuredasanincreaseincellcapacitance)by>80%,whereasinhibitionofL-typeCa(2+)channelsonlyhadaminoreffect.
CONCLUSIONS:
Voltage-gatedT-typeandL-typeCa(2+)channelsaswellasNa(+)channelsparticipateinglucose-stimulatedelectricalactivityandinsulinsecretion.Ca(2+)-activatedBKchannelsarerequiredforrapidmembranerepolarization.Exocytosisofinsulin-containinggranulesisprincipallytriggeredbyCa(2+)influxthroughP/Q-typeCa(2+)channels.
BraunM.,etal.(2008) Voltage-gatedionchannelsinhumanpancreaticbeta-cells:electrophysiologicalcharacterizationandroleininsulinsecretion.Diabetes.PMID:18390794
Structuralbasisofbindingandinhibitionofnoveltarantulatoxinsinmammalianvoltage-dependentpotassiumchannels
Voltage-dependentpotassiumchannelKv2.1iswidelyexpressedinmammalianneuronsandwassuggestedresponsibleformediatingthedelayedrectifier(I(K))currents.Furtherinvestigationofthecentralroleofthischannelrequiresthedevelopmentofspecificpharmacology,forinstance,theutilizationofspidervenomtoxins.Mostofthesetoxinsbelongtothesamestructuralfamilywithashortpeptidereticulatedbydisulfidebridgesandshareasimilarmodeofaction.Hanatoxin1(HaTx1)fromaChileantarantulawasoneoftheearliestdiscussedtoolsregardingthisandhasbeenintensivelyappliedtocharacterizethechannelblockingnotthroughtheporedomain.Recently,morerelatednoveltoxinsfromAfricantarantulassuchasheteroscordratoxins(HmTx)andstromatoxin1(ScTx1)wereisolatedandshowntoactasgatingmodifierssuchasHaTxonKv2.1channelswithelectrophysiologicalrecordings.However,furtherinteractiondetailsareunavailableduetothelackofhigh-resolutionstructuresofvoltage-sensingdomainsinsuchmammalianKvchannels.Therefore,inthepresentstudy,weexploredstructuralobservationviamoleculardockingsimulationbetweentoxinsandKv2.1channelsbaseduponthesolutionstructuresofHaTx1andatheoreticalbasisofanindividualS3(C)helicalchannelfragmentincombinationwithhomologymodelingforothernoveltoxins.Ourresultsprovideprecisechemicaldetailsfortheinteractionsbetweenthesetarantulatoxinsandchannel,reasonablycorrelatingthepreviouslyreportedpharmacologicalpropertiestothethree-dimensionalstructuralinterpretation.Inaddition,itissuggestedthatcertainsubtlestructuralvariationsontheinteractionsurfaceoftoxinsmaydiscriminatebetweentherelatedtoxinswithdifferentaffinitiesforKvchannels.Evolutionarylinksbetweenspiderpeptidetoxinsanda“voltagesensorpaddles”mechanismmostrecentlyfoundinthecrystalstructureofanarchaebacterialK(+)channel,KvAP,arealsodelineatedinthispaper.
SchiauYS., etal. (2003) Structuralbasisofbindingandinhibitionofnoveltarantulatoxinsinmammalianvoltage-dependentpotassiumchannels. ChemResToxicol.PMID:14565763
NovelTarantulaToxinsforSubtypesofVoltage-DependentPotassiumChannelsintheKv2andKv4Subfamilies
Threenovelpeptideswiththeabilitytoinhibitvoltage-dependentpotassiumchannelsintheshab(Kv2)andshal(Kv4)subfamilieswereidentifiedfromthevenomoftheAfricantarantulasStromatopelmacalceata(ScTx1)andHeteroscodramaculata(HmTx1,HmTx2).Thethreetoxinsare34-to38-aminoacidpeptidesthatbelongtothestructuralfamilyofinhibitorcystineknotspiderpeptidesreticulatedbythreedisulfidebridges.ElectrophysiologicalrecordingsinCOScellsshowthatthesetoxinsactasgatingmodifierofvoltage-dependentK+channels.ScTx1isthefirsthigh-affinityinhibitoroftheKv2.2channelsubtype(IC50,21.4nM)tobedescribed.ScTx1alsoinhibitstheKv2.1channels,withanIC50of12.7nM,andKv2.1/Kv9.3heteromultimersthathavebeenproposedtobeinvolvedinO2sensinginpulmonaryarterymyocytes.Inaddition,itisthemosteffectiveinhibitorofKv4.2channelsdescribedthusfar,withanIC50of1.2nM.HmTxtoxinssharesequencesimilaritieswithboththepotassiumchannelblockertoxins(HmTx1)andthecalciumchannelblockertoxinomega-GsTxSIA(HmTx2).TheyinhibitpotassiumcurrentassociatedwithKv2subtypesinthe100to300nMconcentrationrange.HmTx2seemstobeaspecificinhibitorofKv2channels,whereasHmTx1alsoinhibitsKv4channels,includingKv4.1,withthesamepotency.HmTx1isthefirstdescribedpeptideeffectoroftheKv4.1subtype.Thosenoveltoxinsarenewtoolsfortheinvestigationofthephysiologicalroleofthedifferentpotassiumchannelsubunitsincellularphysiology.
EscoubasP.,etal.(2002) NovelTarantulaToxinsforSubtypesofVoltage-DependentPotassiumChannelsintheKv2andKv4Subfamilies.MolPharm.PMID:12065754
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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。
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.
两个CEX方法A和B测定同一单抗，结果碱性峰比例差不多，酸性峰比例相差约7%，相应主峰也差了7%左右。
具体来说，A方法酸性峰高，主峰低，碱性峰稍微低点；B方法酸性峰低，主峰高，碱性峰稍微高点；另外也做了CIEF，结果呢和A方法更接近。
仔细比较起来，AB两个方法的峰性和数量差不多，就不知道为什么会有这么大的差异。两个方法一个用的WCX柱-磷酸缓冲液，一个用SCX柱-MES缓冲液
大家帮我分析下：
1.两个方法哪个方法更准确，是以酸性峰高的为准还是什么？为什么？
2.这显著差异是由方法造成，具体原因是什么？柱子？
3.CIEF的结果和A方法更接近，是不是可以由此证明A方法更好或者CIEF的方法更好（因为CIEF更快更方便）？
欢迎讨论~
纠正下，A方法用的是Tosoh的柱子，B方法用的是SCX柱。TOSOH的柱子是7um的填料，10cm长。SCX是10um的填料。我本人TOSOH的阳离子柱子用的很少，这次信手用用，结果发现差异很大
那我现在就考虑，在以后方法开发过程中，除了通过流动相pH和组成、梯度、柱子选择来获得样品主峰和酸碱性的最大分离，还要关注各峰比例。因为之前比较方法好坏都只看分离度，尤其是主峰和邻近峰的分离度，获得最大分离度，自然可以做到主峰尽可能纯，但从未认真比较过各峰比例。这是一个大疏忽吧！
另外，CIEF和CEX方法原理还是有点差异的，所以分的是不同的异质体，原液放行两个方法肯定是都要做的。问题就是在早期细胞株筛选和工艺开发阶段，哪个方法才是又快又准。CIEF（iCE280)一般15分钟一个样，比CEX快多了。如果CIEF测得主峰要低于CEX结果，是不是真的完全可以取代CEX呢？CEX分离出的峰远比CIEF的多！
欢迎大家继续讨论~
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。
拼音名：Chunhuashui
英文名：PurifiedWater
【性状】本品为无色的澄清液体；无臭，无味。
【检查】酸碱度取本品10ml，加甲基红指示液2滴，不得显红色；另取10ml，加溴麝香草酚蓝指示液5滴，不得显蓝色。氯化物、流酸盐与钙盐取本品，分置三支试管中，每管各50ml。第一管中加硝酸5滴与硝酸银试液1ml，第二管中加氯化钡试液2ml，第三管中加草酸铵试液2ml，均不得发生浑浊。
硝酸盐取本品5ml置试管中，于冰浴中冷却，加10%氯化钾溶液0.4ml与0.1%二苯胺硫酸溶液0.1ml，摇匀，缓缓滴加硫酸5ml，摇匀，将试管子50℃水浴中放置15分钟，溶液产生的蓝色与标准硝酸盐溶液［取硝酸钾0.163g，加水溶解并稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，再精密量取10ml，加水稀释成100ml，摇匀，即得（每1ml相当于1pgNO3）0.3ml，加无硝酸盐的水4.7ml，用同一方法处理后的颜色比较，不得更深（0.000006%）。
亚硝酸盐取本品10ml，置纳氏管中，加对氨基苯磺酰胺的稀盐酸溶液（1→100）lml与盐酸菜乙H肢溶液（0．l＋100）1ml，产生的粉红色，与标准亚硝酸盐溶液〔取亚硝酸钠0.750g（按干燥品计算），加水溶解，稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，摇匀，再精密量取1ml，加水稀释成50ml，摇匀，即得（每1ml相当于1μgNO2）]0.2ml，加无亚硝酸盐的水9.8ml，用同一方法处理后的颜色比较，不得更深（0.000002%）。
氨取本品50ml，加碱性碘化汞钾试液2ml，放置15分钟；如显色，与氯化铵溶液（取氯化铵31.5mg，加无氨水适量使溶解并稀释成1000ml）1.5ml，加元氨水48ml与碱性碘化汞钾试液2ml制成的对照液比较，不得更深（0.00003%）。
二氧化碳取本品25ml，置50ml具塞量筒中，加氢氧化钙试液25ml，密塞振摇，放置，小时内不得发生浑浊。
易氧化物取本品100ml，加稀硫酸10ml，煮沸后，加高锰酸钾滴定液（0.02mol/L）0.10ml，再煮沸10分钟，粉红色不得完全消失。
不挥发物取本品100ml，置105℃恒重的蒸发皿中，在水浴上蒸干，并在105℃干燥至恒重，遗留残渣不得过1mg。
重金属取本品50ml，加水18.5ml，蒸发至20ml，放冷，加醋酸盐缓冲液（pH3.5）2ml与水适量使成25ml，加硫代乙酰胺试液2ml，摇匀，放置2分钟，与标准铅溶液1.5ml加水18.5ml用同一方法处理后的颜色比较，不得更深（0.00003%）。
微生物限度取本品，采用薄膜过滤法处理后，依法检查（附录ⅪJ），细菌、霉菌和酵母菌总数每1ml不得过100个。
【贮藏】密闭保存。
【化学成分】本品为蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的供药用的水，不含任何附加剂。
【分子式与分子量】H2O18.02
【药理作用】溶剂、稀释剂
这里药典纯化水标准中并无PH值项目，请问对纯化水有PH值的要求吗，范围应在多少？请说明出处？
在纯化水检测中，检验酸碱度合格，但是发现PH在8左右。如果按以上标准检验合格，是否要考虑PH值？请知道的解答，谢谢！
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