Guangxitoxin-1E(GxTx-1E) wasisolatedfromthevenomofChilobrachysjingzhao(Chineseearthtigertarantula).Guangxitoxin-1E wasshowntoblock Kv2.1/KCNB1,Kv2.2/KCNB2andKv4.3/KCND3channels withoutsignificanteffectonKv1.2/KCNA2,Kv1.3/KCNA3,Kv1.5/KCNA5,Kv3.2/KCNC2,Cav1.2/CACNA1C,Cav2.2/CACNA1B,Nav1.5/SCN5A,Nav1.7/SCN9AorNav1.8/SCN10Achannels. Guangxitoxin-1E inhibitsKv2.1withanIC50 valueof1nMandKv2.2withanIC50 valueof3nM.BlockofKv4.3occursat10-20foldhigherconcentrations.Guangxitoxin-1E actsasagatingmodifiersinceitshiftsthevoltage-dependenceofKv2.1K+ currentstowardsdepolarizedpotentials.Inpancreaticbeta-cells, Guangxitoxin-1E enhancesglucose-stimulatedinsulinsecretion bybroadeningthecellactionpotentialandenhancingcalciumoscillations.
RecentlyquotedDescription:
AAsequence: Glu-Gly-Glu-Cys4-Gly-Gly-Phe-Trp-Trp-Lys-Cys11-Gly-Ser-Gly-Lys-Pro-Ala-Cys18-Cys19-Pro-Lys-Tyr-Val-Cys24-Ser-Pro-Lys-Trp-Gly-Leu-Cys31-Asn-Phe-Pro-Met-Pro-OH
Presumeddisulfidebridgepattern:Cys4-Cys19,Cys11-Cys24,Cys18-Cys31
Length(aa): 36
Formula: C178H248N44O45S7
MolecularWeight: 3948.70Da
Appearance:Whitelyophilizedsolid
Solubility: waterorsalinebuffer
CASnumber: notavailable
Source: Synthetic
Purityrate: >95%
Reference:
Theroleofvoltage-gatedpotassiumchannelsKv2.1andKv2.2intheregulationofinsulinandsomatostatinreleasefrompancreaticislets
Thevoltage-gatedpotassiumchannelsKv2.1&Kv2.2arehighlyexpressedinpancreaticislets,yettheircontributiontoislethormonesecretionisnotfullyunderstood.HereweinvestigatetheroleofKv2channelsinpancreaticisletsusingacombinationofgenetic&pharmacologicapproaches.Pancreaticβ-cellsfromKv2.1(-/-)micepossessreducedKvcurrent&displaygreaterglucose-stimulatedinsulinsecretion(GSIS)relativetoWTβ-cells.InhibitionofKv2.xchannelswithselectivepeptidyl[guangxitoxin-1E(GxTX-1E)]orsmallmolecule(RY796)inhibitorsenhancesGSISinisolatedwild-type(WT)mouse&humanislets,butnotinisletsfromKv2.1(-/-)mice.However,inWTmiceneitherinhibitorimprovedglucosetoleranceinvivo.GxTX-1E&RY796enhancedsomatostatinreleaseinisolatedhuman&mouseislets&insituperfusedpancreatafromWT&Kv2.1(-/-)mice.Kv2.2silencinginmouseisletsbyadenovirus-smallhairpinRNA(shRNA)specificallyenhancedisletsomatostatin,butnotinsulin,secretion.Inmicelackingsomatostatinreceptor5,GxTX-1Estimulatedinsulinsecretion&improvedglucosetolerance.Collectively,thesedatashowthatKv2.1regulatesinsulinsecretioninβ-cells&Kv2.2modulatessomatostatinreleaseinδ-cells.DevelopmentofselectiveKv2.1inhibitorswithoutcrossinhibitionofKv2.2mayprovidenewavenuestopromoteGSISforthetreatmentoftype2diabetes.
LiXN., etal. (2013)Theroleofvoltage-gatedpotassiumchannelsKv2.1andKv2.2intheregulationofinsulinandsomatostatinreleasefrompancreaticislets. JPharmacolExpTher. PMID: 23161216
Regulationofvoltage-gatedK+channelsbyglucosemetabolisminpancreaticbeta-cells
Regulationofdelayedrectifier-typeK(+)channels(Kv-channels)byglucosewasstudiedinratpancreaticbeta-cells.TheKv-channelcurrentwasincreasedinamplitudesbyincreasingglucoseconcentrationfrom2.8to16.6mM,whileitwasdecreasedby2.8mMglucoseinareversIBLemanner(down-regulation)inbothperforated&conventionalwhole-cellmodes.ThecurrentwasdecreasedbyFCCP,intraPipette0mMATPorAMPPNP.Glyceraldehyde,pyruvicacid,2-ketoisocaproicacid,&10mMMgATPpreventedthedown-regulationinducedby2.8mMorlessglucose.TheresidualcurrentaftertreatmentwithKv2.1-specificblocker,guangxitoxin-1E,wasunchangedbyloweringorincreasingglucoseconcentration.WeconcludethatglucosemetabolismregulatesKv2.1channelsinratsbeta-cellsviaalteringMgATPlevels.
YoshidaM., etal. (2009)Regulationofvoltage-gatedK+channelsbyglucosemetabolisminpancreaticbeta-cells. FEBSLett. PMID: 19500583
AnautomatedelectrophysiologyserumshiftassayforK(V)channels
ThepresenceofseruminBIOLOGicalsamplesoftennegativelyimpactsthequalityofinvitroassays.However,assaystolerantofserumareusefulforassessingtheinvivoavailABIlityofasmallmoleculeforitstarget.Electrophysiologyassaysofionchannelsarenotoriouslysensitivetoserumbecauseoftheirrelianceontheinteractionoftheplasmamembranewitharecordingelectrode.Hereweinvestigatethetoleranceofanautomatedelectrophysiologyassayforavoltage-gatedpotassium(K(V))channeltoserum&purifiedplasmaproteins.Thedelayedrectifierchannel,K(V)2.1,stablyexpressedinChinesehamsterovarycellsproduceslarge,stablecurrentsontheIonWorksQuattroplatform(MDSAnalyticalTechnologies,Sunnyvale,CA),makingitanidealtestcase.K(V)2.1currentsrecordedonthisplatformarehighlyresistanttoserum,allowingrecordingsinashighas33%serum.UsingasetofcompoundsrelatedtotheK(V)channelblocker,4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone,weshowthatshiftsincompoundpotencywithwholeserumorisolatedserumproteinscanbereliablymeasuredwiththisassay.Importantly,thisassayisalsorelativelyinsensitivetoplasma,allowingthecreationofabioassayforinhibitorsofK(V)2.1channelactivity.Hereweshowthatsuchabioassaycanquantifythelevelsofthegatingmodifier,guangxitoxin-1E,inplasmasamplesfrommicedosedwiththepeptide.Thisstudydemonstratestheutilityofusinganautomatedelectrophysiologyplatformformeasuringserumshifts&forbioassaysofionchannelmodulators.
RatliffKS., etal.(2008)AnautomatedelectrophysiologyserumshiftassayforK(V)channels. AssayDrugDevTechnol.PMID: 18471078
Gatingmodifierpeptidesasprobesofpancreaticbeta-cellphysiology
Pancreaticbeta-cellsdepolarizeinresponsetoglucose&firecalcium-dependentactionspotentialsthattriggerinsulinsecretion.Themajorcurrentresponsibleforactionpotentialrepolarizationinthesecellsisadelayedrectifier&Kv2.1subunitsarethoughtbeamajorcontributorofthedelayedrectifierchannels.Hence,blockersofKv2.1channelsmightprolongactionpotentials&enhancecalciuminflux&insulinsecretion.However,thelackofspecificsmallmoleculeKv2.1inhibitorshashinderedthetestingofthismechanism.Importantly,severalgatingmodifierpeptidesinhibitKv2.1channelsinarelativelyspecificfashion.Hanatoxin(HaTX)&guangxitoxin-1(GxTX-1)areexamplesthathavebeenusedtoprobetheroleofKv2.1channelsinbeta-cellphysiology.BothHaTX&GxTX-1stronglyinhibittheKvcurrentofbeta-cellsfromvariousspecies,arguingthatKv2.1subunitscontributesignificantlytothebeta-celldelayedrectifier.GxTX-1prolongsglucose-triggeredactionpotentials,enhancesglucose-dependentintracellularcalciumelevations&augmentsglucose-dependentinsulinsecretion.Takentogether,thesedatasuggestthatblockersofKv2.1channelsmaybeausefulapproachtothedesignofnoveltherapeuticagentsforthetreatmentoftype2diabetes.Thesestudieshighlighttheutilityofgatingmodifierpeptidesinthestudyofphysiologicalsystems.
HerringtonJ.,(2009)Gatingmodifierpeptidesasprobesofpancreaticbeta-cellphysiology. Toxicon. PMID: 17101164
SNAP-25(1-180)enhancesinsulinsecretionbyblockingKv2.1channelsinratpancreaticisletbeta-cells
Voltage-gatedoutwardK(+)currentsfrompancreaticisletbeta-cellsareknowntorepolarizetheactionpotentialduringaglucosestimulus,&consequentlytomodulateCa(2+)entry&insulinsecretion.ThevoltagegatedK(+)(Kv)channel,Kv2.1,whichisexpressedinratisletbeta-cells,mediatesover60%oftheKvoutwardK(+)currents.AnovelpeptidylinhibitorofKv2.1/Kv2.2channels,guangxitoxin(GxTX)-1,hasbeenshowntoenhanceglucose-stimulatedinsulinsecretion.Here,weshowthatSNAP-25(1-180)(S180),anN-terminalSNAP-25domain,butnotSNAP-25(1-206)(S206),inhibitsKvcurrent&enhancesglucose-dependentinsulinsecretionfromratpancreaticisletbeta-cells,&furThermore,thisenhancementwasinducedbytheblockadeoftheKv2.1current.ThisstudyindicatesthattheKv2.1channelisapotentialtargetfornoveltherapeuticagentdesignforthetreatmentoftype2diabetes.Thistargetmaypossessadvantagesovercurrently-usedtherapies,whichmodulateinsulinsecretioninaglucose-independentmanner.
ZhuangGQ., etal. (2009)SNAP-25(1-180)enhancesinsulinsecretionbyblockingKv2.1channelsinratpancreaticisletbeta-cells. BiochemBiophysResCommun. PMID: 19103161
Blockersofthedelayed-rectifierpotassiumcurrentinpancreaticbeta-cellsenhanceglucose-dependentinsulinsecretion
Delayed-rectifierK+currents(I(DR))inpancreaticbeta-cellsarethoughttocontributetoactionpotentialrepolarization&therebymodulateinsulinsecretion.Thevoltage-gatedK+channel,K(V)2.1,isexpressedinbeta-cells,&thebiophysicalcharacteristicsofheterologouslyexpressedchannelsaresimilartothoseofI(DR)inrodentbeta-cells.AnovelpeptidylinhibitorofK(V)2.1/K(V)2.2channels,guangxitoxin(GxTX)-1(half-maximalconcentrationapproximately1nmol/l),hasbeenpurified,characterized,&usedtoprobethecontributionofthesechannelstobeta-cellphysiology.Inmousebeta-cells,GxTX-1inhibits90%ofI(DR)&,asforK(V)2.1,shiftsthevoltagedependenceofchannelactivationtomoredepolarizedpotentials,acharacteristicofgating-modifierpeptides.GxTX-1broadensthebeta-cellactionpotential,enhancesglucose-stimulatedintracellularcalciumoscillations,enhancesinsulinsecretionfrommousepancreaticisletsinaglucose-dependentmanner.Thesedatapointtoamechanismforspecificenhancementofglucose-dependentinsulinsecretionbyapplyingblockersofthebeta-cellI(DR),whichmayprovideadvantagesovercurrentlyusedtherapiesforthetreatmentoftype2diabetes.
HerringtonJ., etal.(2006)Blockersofthedelayed-rectifierpotassiumcurrentinpancreaticbeta-cellsenhanceglucose-dependentinsulinsecretion. Diabetes. PMID: 16567526
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