GsMTx4(GsMTx-4,M-theraphotoxin-Gr1a) hasbeenisolatedfromthevenomofthespiderGrammostolarosea.Thiscationichydrophobicpolypeptideblocksselectivelythegatingofcationselectivechannelsandmechanosensitiveionchannelssuchas TRPC1 or TRPC6,withouthavinganyeffectonwhole-cellvoltage-sensitivecurrents.Thistoxinactsbyperturbingtheinterfacebetweenthechannelandthelipidbilayerwithoutnecessarilybeinginphysicalcontactwiththechannel. GsMTx4 alsodemonstratedtoactive TRPA1 channelat1µMconcentrationandtoinhibit Piezo1currents.Recentlyquoted Description:Productcode:N/A.Categories:Mechanosensitivechannels,TRPchannels.Tags:mechanosensitive,stretch,trpc.AAsequence: Gly-Cys2-Leu-Glu-Phe-Trp-Trp-Lys-Cys9-Asn-Pro-Asn-Asp-Asp-Lys-Cys16-Cys17-Arg-Pro-Lys-Leu-Lys-Cys23-Ser-Lys-Leu-Phe-Lys-Leu-Cys30-Asn-Phe-Ser-Phe-NH2Disulfidebonds: Cys2-Cys17,Cys9-Cys23 andCys16-Cys30Length(AA): 34Formula: C185H273N49O45S6MolecularWeight: 4095.2DaAppearance: WhitelyophilizedsolidSolubility: waterandsalinebufferCASnumber:Source: SyntheticPurityrate: >97%Reference:CitationsAndersonM,etal.(2013)OpposingeffectsofpodocinonthegatingofpodocyteTRPC6channelsevokedbymembranestretchordiacylglycerol. AmJPhysiolCellPhysiol. PubMedlinkSynergybetweenPiezo1andPiezo2channelsconfershigh-strainmechanosensitivitytoarticularcartilageDiarthrodialjointsareessentialforloadbearingandlocomotion.Physiologically,articularcartilagesustainsmillionsofcyclesofmechanicalloADIng.Chondrocytes,thecellsincartilage,regulatetheirmetabolicactivitiesinresponsetomechanicalloading.Pathologicalmechanicalstresscanleadtomaladaptivecellularresponsesandsubsequentcartilagedegeneration.Wesoughttodeconstructchondrocytemechanotransductionbyidentifyingmechanosensitiveionchannelsfunctioningatinjuriouslevelsofstrain.Wedetectedrobustexpressionoftherecentlyidentifiedmechanosensitivechannels,PIEZO1andPIEZO2.CombineddirectedexpressionofPiezo1and-2sustainedpotentiatedmechanicallyinducedCa(2+)signalsandelectricalcurrentscomparedwithsingle-Piezoexpression.Inprimaryarticularchondrocytes,mechanicallyevokedCa(2+)transientsproducedbyatomicforcemicroscopywereinhibitedbyGsMTx4,aPIEZO-blockingpeptide,andbyPiezo1-orPiezo2-specificsiRNA.Wecomplementedthecellularapproachwithanexplant-cartilageinjurymodel.GsMTx4reducedchondrocytedeathaftermechanicalinjury,suggestingapossIBLetherapyforreducingcartilageinjuryandposttraumaticosteoarthritisbyattenuatingPiezo-mediatedcartilagemechanotransductionofinjuriousstrains.LeeW,etal.(2015)SynergybetweenPiezo1andPiezo2channelsconfershigh-strainmechanosensitivitytoarticularcartilage.PNAS. PubMedlinkTheMechanosensitiveIonChannelPiezo1IsInhibitedbythePeptideGsMTx4Cellscanrespondtomechanicalstressbygating mechanosensitive ionchannels (MSCs).Thecloningof Piezo1,aeukaryoticcationselectiveMSC,definesanewsystemforstudyingmechanicaltransductionatthecellularlevel.Because Piezo1 haselectrophysiologicalpropertiessimilartothoseofendogenouscationicMSCsthatareselectively inhibited bythe peptide GsMTx4,wetestedwhetherthe peptide targets Piezo1 activity.ExtracellularGsMTx4 atmicromolarconcentrationsreversibly inhibited ∼80%ofthemechanicallyinducedcurrentofoutside-outpatchesfromtransfectedHEK293cells.Theinhibitionwasvoltageinsensitive,&asseenwithendogenousMSCs,themirrorimagedenantiomer inhibited likethel.Therateconstantsforbinding&unbindingbasedon Piezo1 currentkineticsprovidedassociation&dissociationratesof7.0×10(5)M(-1)s(-1)&0.11s(-1),respectively,&aK(D)of∼155nM,similartovaluespreviouslyreportedforendogenousMSCs.Consistentwithpredictedgatingmodifierbehavior,GsMTx4 producedan∼30mmHgrightwardshiftinthepressure-gatingcurve&wasactiveonclosed channels.Incontrast,streptomycin,anonspecificinhibitorofcationicMSCs,showedtheuse-dependentinhibitioncharacteristicofopen channel block.The peptide didnotblockcurrentsofthemechanical channel TREK-1onoutside-outpatches.Whole-cell Piezo1 currentswerealsoreversibly inhibited by GsMTx4,&althoughtheoffratewasnearlyidenticaltothatofoutside-outpatches,differenceswereobservedfortheonrate.TheABIlityof GsMTx4 totargetthemechanosensitivityof Piezo1 supportstheuseofthis channel inhigh-throughputscreensforpharmacologicalagents&diagnosticassays.Bae,C.,  etal. (2011)TheMechanosensitiveIonChannelPiezo1IsInhibitedbythePeptideGsMTx4.Biochemistry. PMID: 21696149TRPA1IsDifferentiallyModulatedbytheAmphipathicMoleculesTrinitrophenolandChlorpromazineTRPA1,apoorlyselectiveCa(2+)-permeablecationchannel,isexpressedinperipheralsensoryneurons,whereitisconsideredtocontributetoavarietyofsensoryprocessessuchasthedetectionofpainfulstimuli.FurThermore,TRPA1wasalsoidentifiedinhaircellsoftheinnerear,butitsinvolvementinsensingmechanicalforcesisstillbeingcontroversiallydiscussed.Amphipathicmoleculessuchastrinitrophenol&chlorpromazinehavebeenshowntoprovideusefultoolstostudymechanosensitivechannels.Dependingontheircharge,theypartitionintheinneroroutersheetsofthelipidbilayer,causingacurvatureofthemembrane,whichhasbeendemonstratedtoactivateorinhibitmechanosensitiveionchannels.Inthepresentstudy,weinvestigatedtheeffectofthesemoleculesonTRPA1gating.TRPA1wasrobustlyactivatedbytheanionicamphipathicmoleculetrinitrophenol.Thewhole-cell&singlechannelpropertiesresemblethosepreviouslydescribedforTRPA1.Moreover,wecouldshowthatthetoxinGsMTx-4actsonTRPA1.Inadditiontoitsrecentlydescribedroleasaninhibitorofstretch-activatedionchannels,itservesasapotentactivatorofTRPA1channels.Ontheotherhand,thepositivelychargeddrugchlorpromazinemodulatesactivatedTRPA1currentsinavoltage-dependentway.TheexposureofactivatedTRPA1channelstochlorpromazineledtoablockatpositivepotentials&anincreasedopenprobabilityatnegativepotentials.ThevariabilityintheshapeoftheI-VcurvegivesafirstindicationthatnativemechanicallyactivatedTRPA1currentsmustnotnecessarilyexhibitthesamebiophysicalpropertiesasligand-activatedTRPA1currents.KerstinHill,MichaelSchaefer(2007)TRPA1IsDifferentiallyModulatedbytheAmphipathicMoleculesTrinitrophenolandChlorpromazine. JBC. PMID: 17218316Moleculardynamicssimulationsofastretch-activatedchannelinhibitorGsMTx4withlipidmembranes:twobindingmodesandeffectsoflipidstructureOurrecentmoleculardynamicssimulationstudyofhanatoxin1(HaTx1),agatingmodifierthatbindstothevoltagesensorofK(+)channels,hasshownthatHaTx1hastheabilitytointeractwithcarbonyloxygenatomsofbothleafletsofthelipidbilayermembrane&tobelocatedatadeeppositionwithinthemembrane.HereweperformedasimilarstudyofGsMTx4,astretch-activatedchannelsinhibitor,belongingtothesamepeptidefamilyasHaTx1.Bothtoxinshaveanellipsoidalshape,abeltofpositivelychargedresiduesaroundtheperiphery,&ahydrophobicprotrusion.Resultsshowthat,likeHaTx1,GsMTx4caninteractwiththemembraneintwodifferentways.Whenallthepositivelychargedresiduesinteractwiththeouterleafletlipid,GsMTx4canassumeashallowbindingmode.Ontheotherhand,whentheelectrostaticinteractionbringsthepositivelychargedgroupsofK-8&K-28intothevicinityofthecarbonyloxygenatomsoftheinnerleafletlipids,thesystemexhibitsadeepbindingmode.Thisdeepmodeisaccompaniedbylocalmembranethinning.ForbothHaTx1&GsMTx4,ourmeanforcemeasurementanalysesshowthatthedeepbindingmodeisenergeticallyfavoredovertheshallowmodewhenaDPPC(dipalmitoyl-phosphatidylcholine)membraneisusedat310K.Incontrast,whenaPOPC(palmitooleoyl-phosphatidylcholine)membraneisusedat310K,thetwobindingmodesexhibitedsimilarstabilityforbothtoxins.SimilaranalyseswithDPPCmembraneat330Kledtoanintermediaryresultbetweentheabovetworesults.Therefore,thestructureofthelipidacylchainsappearstoinfluencethelocation&thedynamicsofthetoxinswithinBIOLOGicalmembranes.Wealsocomparedthebehaviorofanarginine&alysineresiduewithinthemembrane.ThisisofinterestbecausethearginineresidueinteractionwiththelipidcarbonyloxygenatomsmediatesthedeepbindingmodeforHaTx1,whereasthelysineresidueplaysthatroleforGsMTx4.Thearginineresiduegenerallyshowssmootherdynamicsnearthelipidcarbonyloxygenatomsthanthelysineresidue.Thisdifferencebetweenarginine&lysinemaypartlyaccountforthefunctionaldiversityofthemembersofthetoxinfamily.NishizawaM,NishizawaK.(2007)Moleculardynamicssimulationsofastretch-activatedchannelinhibitorGsMTx4withlipidmembranes:twobindingmodesandeffectsoflipidstructure.BiophysJ. PMID: 17384064Localizationofthevoltage-sensortoxinreceptoronKvAPAvarietyofvenomousanimalsproducesmallproteintoxinsthatimpairthefunctionofvoltage-dependentcationchannelsbyaffectingthemotionsofthevoltage-sensordomains&alteringtheenergeticsoftheopeningofthechannel.Inthisstudy,weinvestigatethelocationofthereceptorfortarantulavenomvoltage-sensortoxinsonthevoltage-dependentK+channelfromAeropyrumpernix(KvAP),anarcheabacterialchannelthatisfunctionallyinhibitedbymembersofthistoxinfamily.WeshowthatitispossibletopurifythesamesetoftoxinsfromvenomofthetarantulaGrammostolaspatulatausingeitherthepurifiedKvAPvoltage-sensordomainorthefull-lengthKvAPchannel.Theequivalenceoftoxinretentionprofilesforthetwochannelproteinsimpliesthatthetarantulavoltage-sensortoxinreceptorresidesexclusivelyonthevoltage-sensordomain&thattheporeisnotrequiredforthetoxin-channelinteraction.Wehaveidentified&characterizedthefunctionalpropertiesofasubsetofthetarantulatoxinsthatbindtotheKvAPvoltage-sensordomain.Someofthesetoxins,VSTX1&GSMTX4,havebeenpreviouslyisolated,whileothers,VSTX2&VSTX3,arenewmembersofthetarantulavoltage-sensortoxinfamily.Somebutnotalltoxinsthatbindtothevoltage-sensordomainaffectvoltage-dependentgatingofKvAPchannelsinlipidmembranes.Ruta,V.,andMacKinnon,R.(2004)Localizationofthevoltage-sensortoxinreceptoronKvAP,Biochemistry. PMID: 15287735CDNAsequenceandinvitrofoldingofGsMTx4,aspecificpeptideinhibitorofmechanosensitivechannelsThepeptideGsMTx4fromthetarantulavenom(Grammostolaspatulata)inhibitsmechanosensitiveionchannels.Inthiswork,wereportthecDNAsequenceencodingGsMTx4.Thegeneistranslatedasaprecursorproteinof80aminoacids.Thefirst21aminoacidsareapredictedsignalsequence&theC-terminalresiduesareasignalforamidation.AnarginineresidueadjacenttotheN-terminalglycineofGsMTx4isthecleavagesiteforrelease.Theresultingpeptideis34aminoacidsinlengthwithaC-terminalphenylalanine&notaserine-alaninepreviouslyidentified[J.Gen.Physiol.115(2000)583].Wechemicallysynthesizedthispeptide&foldeditin0.1MTris,pH7.9withoxidized/reducedglutathione(1/10).Propertiesofthesyntheticpeptidewereidenticaltothewildtypeforhighperformanceliquidchromatography(HPLC),massspectrometry,CD,&NMR.WealsoclonedGsMTx4inathioredoxinfusionproteinsystemcontainingsixhistidines.Nickelaffinitycolumnsallowedrapidpurification&foldingoccurredinconditionsdescribedabovewith0.5MguanidiniumHClpresent.ThrombincleavageliberatedGsMTx4withthreeextraaminoacidsattheN-terminus.TheretentiontimeinHPLCanalysis&theCDspectrumwassimilartowildtype.Boththesynthetic&clonedpeptideswereactiveinthepatchclampassay.Ostrow,K.L., etal. (2003)cDNAsequenceandinvitrofoldingofGsMTx4,aspecificpeptideinhibitorofmechanosensitivechannels. Toxicon. PMID: 14559077SolutionstructureofpeptidetoxinsthatblockmechanosensitiveionchannelsMechanosensitivechannels(MSCs)playkeyrolesinsensoryprocessing&havebeenimplicatedasprimarytransducersforavarietyofcellularresponsesrangingfromosmosensingtogeneexpression.ThispaperpresentsthefirststructuresofanykindknowntointeractspecificallywithMSCs.GsMTx-4&GsMtx-2areinhibitorcysteineknotpeptidesisolatedfromvenomofthetarantula,Grammostolaspatulata(Suchyna,T.M.,Johnson,J.H.,Hamer,K.,Leykam,J.F.,Gage,D.A.,Clemo,H.F.,Baumgarten,C.M.,&Sachs,F.(2000)J.Gen.Physiol.115,583-598).InhibitionofcationicMSCsbythehigheraffinityGsMtx-4(K(D)approximately500nm)reducedcellsizeinswollen&hypertrophicheartcells,swelling-activatedcurrentsinastrocytes,&stretch-inducedarrhythmiasintheheart.Despitetherelativelylowaffinity,nocross-reactivityhasbeenfoundwithotherchannels.Usingtwo-dimensionalNMRspectroscopy,wedeterminedthesolutionstructureofGsMTx-4&aloweraffinity(GsMTx-2;K(D)approximately6microm)peptidefromthesamevenom.Thedominantfeatureofthetwostructuresisahydrophobicpatch,utilizingmostofthearomaticresidues&surroundedwithchargedresidues.ThespatialarrangementofchargedresiduesthatareuniquetoGsMTx-4&GsMTx-2mayunderlietheselectivityofthesepeptides.Oswald,R.E., etal. (2002)Solutionstructureofpeptidetoxinsthatblockmechanosensitiveionchannels. JBiolChem. PMID: 12082099IdentificationofapeptidetoxinfromGrammostolaspatulataspidervenomthatblockscation-selectivestretch-activatedchannelsWehaveidentifieda35aminoacidpeptidetoxinoftheinhibitorcysteineknotfamilythatblockscationicstretch-activatedionchannels.Thetoxin,denotedGsMTx-4,wasisolatedfromthevenomofthespiderGrammostolaspatulata&has<50%homologytootherneuroactivepeptides.ItwasisolatedbyfractionatingwholevenomusingreversephaseHPLC,&thenassayingfractionsonstretch-activatedchannels(SACs)inoutside-outpatchesfromadultratastrocytes.Althoughthechannelgatingkineticsweredifferentbetweencell-attached&outside-outpatches,thepropertiesassociatedwiththechannelpore,suchasselectivityforalkalications,conductance(approximately45pSat-100mV)&amildrectificationwereunaffectedbyoutside-outformation.GsMTx-4producedacompleteblockofSACsinoutside-outpatches&appearedspecificsinceithadnoeffectonwhole-cellvoltage-sensitivecurrents.Theequilibriumdissociationconstantofapproximately630nMwascalculatedfromtheratioofassociationanddissociationrateconstants.Inhypotonicallyswollenastrocytes,GsMTx-4producesapproximately40%reductioninswelling-activatedwhole-cellcurrent.Similarly,inisolatedventricularcellsfromarabbitdilatedcardiomyopathymodel,GsMTx-4producedanearcompleteblockofthevolume-sensitivecation-selectivecurrent,butdidnotaffecttheanioncurrent.Inthemyopathicheartcells,wheretheswell-inducedcurrentistonicallyactive,GsMTx-4alsoreducedthecellsize.Thisisthefirstreportofapeptidetoxinthatspecificallyblocksstretch-activatedcurrents.Thetoxinaffectonswelling-activatedwhole-cellcurrentsimplicatesSACsinvolumeregulation.Suchyna,T.M., etal. (2000)IdentificationofapeptidetoxinfromGrammostolaspatulataspidervenomthatblockscation-selectivestretch-activatedchannels. JGenPhysiol. PMID: 10779316