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HeterogeneityinhumanhippocampalCaMKIItranscriptsrevealsanallostericroleforthehubdomaininactivityregulationRomanSloutsky*1,NoelleDziedzic*1,2,MatthewJ.Dunn1,RachelM.Bates1,AnaP.Torres-Ocampo1,2,BrendanPage1,JohnG.Weeks1,MargaretM.Stratton1DepartmentofBiochemistryandMolecularBiology1,MolecularandCellularBiologyGraduateProgram2,UniversityofMassachusetts,Amherst,UnitedStates*TheseauthorscontributedequallyABSTRACTCa2+-calmodulindependentproteinkinaseII(CaMKII)playsacentralroleinCa2+signalingthroughoutthebody.Specificallyinthehippocampus,CaMKIIisrequiredforlearningandmemory.CaMKIIisencodedbyfourhighlyconservedgenesinvertebrates:α,β,γ,andδ.AllCaMKIIsarecomprisedofakinasedomain,regulatorysegment,variablelinkerregion,andhubdomainresponsibleforoligomerization.Thefourgenesdifferprimarilyinlinkerlengthandcompositionduetoextensivealternativesplicing.Numerousquestionsregardingthesesplicevariantsstillremain,startingwiththefundamentalquestionofwhichvariantsareexpressedandhowsplicingaffectsCaMKIIregulation.Here,weunambiguouslyreporttheheterogeneityofCaMKIItranscriptsinacomplexsampleofhumanhippocampususingIlluminasequencing.Ourresultsshowthathippocampalneuronscontainadiversecollectionof56CaMKIItranscriptsfromallfourCaMKIIgenes,including25thathavenotbeenpreviouslyannotated.WecharacterizedtheCa2+/CaMsensitivityofhippocampalCaMKIIvariantsspanningabroadrangeoflinkerlengthsandcompositions.WedemonstratethattheeffectofthevariablelinkeronCa2+/CaMsensitivityisconditionalonkinaseandhubdomains.Moreover,werevealanovelroleforthehubdomainasanallostericregulatorofkinaseactivity,whichmayprovideanewpharmacologicaltargetformodulatingCaMKIIactivity.Takentogether,weproposethatCa2+/CaMsensitivityinCaMKIIisgene-dependentandincludessignificantcontributionsfromthehub.OursequencingapproachcombinedwithbiochemistryprovidesnewinsightsintounderstandingthecomplexpoolofendogenousCaMKII.SIGNIFICANCECaMKIIisawell-conservedproteinthatisessentialforlearningandmemory.WhenCaMKIIismutatedinamouse,thismousehasdifficultylearningandrememberinghowtogetthroughamaze.Thehippocampusisthepartofthebrainrequiredformemory.Here,weusedaspecificexperimenttodetermineeverytypeofCaMKIIthatisinahumanhippocampus.Wefound56differenttypesandthenaskedhowthesedifferencesaffectCaMKIIfunction.ThesedataprovideevidencethatanunexpecteddomainofCaMKIIisimportantforregulatingitsactivity.ThisnewfindinghelpsusbetterunderstandendogenousCaMKIIinthebrainandprovidesanewmechanismformodulatingCaMKIIactivity.

certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted August 1, 2019. . https://doi.org/10.1101/721589doi: bioRxiv preprint

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INTRODUCTIONCa2+/calmodulindependentproteinkinaseII(CaMKII)isacrucialoligomericSer/Thrkinaseinlong-termmemoryformation(1)aswellaseggactivationinfertilization(2,3),andcardiacpacemaking(4).CaMKIIisexpressedthroughoutthehumanbody,whichfacilitatesitsroleinthesedisparatefunctions.TherearefourhumangenesthatencodeCaMKII:CaMKIIαandβ(predominantlyinbrain),CaMKIIδ(predominantinheart)andCaMKIIγ(multipleorgansystems,includingeggandsperm)(5-7).Inallfourgenes,eachCaMKIIsubunitiscomprisedofakinasedomain,regulatorysegment,variablelinkerregion,andahubdomain(Fig.1A).Thekinaseandhubdomainsofallfourgenesarehighlyconservedwithminimum90%and75%pairwiseidentity,respectively.Thelinkerthatconnectsthekinaseandhubdomainsishighlyvariableinlengthandcompositionduetoalternativesplicing.Herein,wefocusonCaMKIIinthehippocampus,whichcomprisestheprimarymemorycenterinthebrain(8).CaMKIIisthemostabundantenzymeinneuronaldendritesandhasbeenimplicatedfunctionallyinmemory(1).TransgenicmicedeficientinneuronalCaMKIIhavelimitedlong-termmemoryanddisplayspecificlearningimpairments(9,10).Morespecifically,micewithmutationsatcriticalphosphorylationsitesinCaMKIIdisplaydefectsinhigherordermemoryandadaptivelearning(11).ThestructureandfunctionofboththekinaseandhubdomainsofCaMKIIhavebeenwellstudied.Kinaseactivityiscontrolledbytheautoinhibitoryregulatorysegment,whichblocksthesubstrate-bindingsiteintheabsenceofCa2+(12).WhenCa2+levelsrise,Ca2+boundcalmodulin(Ca2+/CaM)competitivelybindstotheregulatorysegmentandrelievesinhibitionbyexposingthesubstrate-bindingsite(Fig.1B)(13).Theregulatorysegmentalsohousesthreesitesofautophosphorylation:Thr286,Thr305,Thr306(followingCaMKIIαnumbering)(14).Notably,phosphorylationatThr286resultsinautonomousactivation,or,sustainedactivityintheabsenceofCa2+(15).Thehubdomainoligomerizestheholoenzymeintobothdodecamericandtetradecamericassemblies(Fig.1C)(16-18). Thevariablelinkerregionhasbeendifficulttostudyforseveralreasons.Thesequenceofthelinkerispredictedtobedisordered.Itislikelyforthisreasonthathigh-resolutionstructuresoftheCaMKIIholoenzymeincludingthelinkerregionhavenotbeensolved.Indeed,theonlyatomicresolutionstructureofaCaMKIIholoenzymeisoneinwhichthevariablelinkeriscompletelydeleted(19).Inthisstructure,theregulatorysegmentmakesextensivecontactswiththehubdomain,therebycompletelysequesteringtheCaMbindingsegmentfromCa2+/CaMinthiscompactconformation.Thisstructure,combinedwithbiochemicaldatadiscussedbelow,ledtothehypothesisthatlinkerlengthmaytunetheautoinhibitionwithintheholoenzymebyeitherstabilizingacompactconformation(shortlinkerforms)orpopulatinganextendedconformationwheretheCaMbindingsegmentismoreaccessible(longerlinkerforms).ThevariablelinkeralsovariesincompositionbetweenthefourCaMKIIgenes.Furthercomplexityisgeneratedfromalternativesplicingupto9exons(ex13-21)ineachlinker,creatingatotalofmorethan50variantspossiblyexpressedfromfourgenes(Fig.2A).Thecombinationofhighidentitywithintheconservedkinaseandhubdomainsandhighvariability


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inthelinkerregionmakesitdifficulttospecificallyidentifysplicevariantsofparalogsincellularexperiments.Therehavebeenmanystudiesthatoutlinespecificrolesforexonsinthevariablelinkerregion,whichprovidesomecluesastowhyexonsmaybesplicedinorout.Exon15,whichisencodedinallgeneswiththeexceptionofCaMKIIβhasbeendocumentedtocontainanuclearlocalizationsequence(NLS)(20-22).ThisNLS(KKRK)isacommonmotifidentifiedasasufficientsequencetoachievenuclearlocalization(23).Exon13,whichisencodedinCaMKIIβandγ,butnotαorδ,mediatesanimportantinteractionwithF-actin(24,25).Indendriticspines,CaMKIIinteractingwithactiniscrucialforthemaintenanceofspinestructureduringlong-termpotentiation(26).PerhapsthemostdramaticformofalternativesplicingresultsintheformationofalphaCaMKIIassociatedprotein(alphaKAP)whereCaMKIIαissplicedtoaformthatincludesonlythehubdomain(27).ThissplicevariantplaysanimportantroleinmembraneanchoringusingitshydrophobicN-terminaldomain(28).Finally,itwasrecentlyshownthatalternativesplicingwithinthehubmightregulateoligomerization(5).Rolesfortheremainingexonshaveyettobedelineated.Inadditiontorolesassignedtospecificexons,thevariablelinkerregionhasalsobeenshowntoplayacriticalroleinCaMKIIactivation.AstandardmetricforCaMKIIactivityistheconcentrationofCa2+/CaMrequiredforhalf-maximalactivation(EC50value)(19,29).IthasalsobeenclearlyshownthatCaMKIIactivationiscooperative(29).Inregardtolinkerlength,aCaMKIIαvariantwitha30-residuelinkerhasbeenshowntorequiresignificantlylessCa2+/CaMtoachievemaximalactivitycomparedtoaCaMKIIαvariantwitha0-residuelinker(19).ThisgeneraltrendisalsotruewhencomparingalonglinkerCaMKIIβvarianttoashortlinkerCaMKIIαvariant(30).Here,weaddressthegapsinknowledgeintwomajorareas:1)whichspecificCaMKIIsplicevariantsareexpressedinhumanhippocampalcells,and2)howCaMKIIactivationisregulatedwithintheholoenzymestructureforotherCaMKIIgenes.RESULTSTheexonarchitectureisconservedbetweenCaMKIIparalogsThefourgenesencodingCaMKIIinhumansshareahighdegreeofhomology.UsingtheNeighborJoiningmethod(31,32),wecreatedreconstructionsofthekinaseandhubdomaindivergenceforthefourhumanCaMKIIgenesrootedwithD.melanogasterCaMKIIkinaseandhuboutgroups(Fig.1D).Becauseoftheclearone-to-onecorrespondenceofexonsacrossgenes,weemployacommonexonnumberingsystem(Fig.2A).Herein,weimplementanewnamingschemeforCaMKIIvariantsbasedongeneidentityandincorporatedexons(Fig.2C).Movingforward,weproposethisisthesimplestwaytoefficientlycatalogcurrentCaMKIIvariantsandincludenewvariantsastheyarediscovered.Deepsequencingrevealsatleast56CaMKIItranscriptvariantsinhumanhippocampusWeusedIlluminasequencingtounambiguouslydeterminetheCaMKIItranscriptspresentinahumanhippocampussample.


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StrategyofamplificationanddetectionofspecificCaMKIIvariantsInordertoidentifyallalternativelysplicedCaMKIItranscriptsexpressedinahumantotalRNAsample,thesetranscriptsmustbedramaticallyenriched.Similartoearlierapproaches(1,3,(33-35),wereverse-transcribedhumanhippocampalRNAtogeneratecDNAandthenusedgene-specificprimerpairstoPCR-amplifyvariableregionsofCaMKIItranscripts(Fig.2B).AgarosegelseparationofPCRampliconsfollowedbySangersequencingofexcisedgelbandswasinadequatefordetailedcharacterizationofalternativeCaMKIIsplicing,becausemanybandscontainedmixturesofvariants.Tocircumventthisissue,wedevisedanIlluminanext-generationsequencingapproach.GiventhelocationsatwhichCaMKIItranscriptsarevariablysplicedandthemaximallengthsofvariableregionswhenalloptionalexonsareincorporated,strategicplacementofaprimerpairfacilitatesPCR-basedconstructionofIlluminasequencinglibrariessuchthata251bppaired-endsequencingstrategyallowsunambiguousidentificationofvirtuallyallpossibleCaMKIItranscriptsfromallfourgenes(Fig.2B,blue/greenprimerpair).Comparedtotraditionalwhole-transcriptomeIlluminaRNA-Seqlibrarypreparation,thisapproachemphasizessensitivityofvariantdetectionoveraccuracyinquantificationofrelativetranscriptabundance,duetothegreaterpotentialforamplificationbiasresultingfromadditionalPCRcycles.Therefore,wewillnotdiscusstranscriptabundance,excepttosaythateachidentifiedvariantissupportedbyfartoomanyreads(50ormore)tobeattributabletosequencingerrors.AnalysisofIlluminasequencingWedetectedasurprisinglylargenumberofdistincttranscriptvariants(Fig.2C,Supplement1,2),including3variantsofCaMKIIα,24ofCaMKIIβ,17ofCaMKIIγ,and12ofCaMKIIδ.Inadditiontovariantspreviouslyannotatedingenomicdatabases(NCBIConsensusCDScollection,NCBIGenBank,Ensembl),wealsoidentifiednumerouspreviouslyunobservedvariantsofeachgene,whichwerefertoasnovelinthismanuscript.OnenovelvariantofnotewastheCaMKIIαtranscriptincludingnooptionallinkerexons(0aalinker).ThisissignificantbecausethecrystalstructureofCaMKIIαno-linkervariantistheonlyavailablestructureoffulllengthCaMKII(PDB:3SOA(19)),butthisvariantwaspreviouslynotknowntobebiologicallyrelevant.Nearlyeveryidentifiedtranscriptincorporatingoptionallinkerexonscontainedexons14and18,suggestingthesetwoexonsconstitutea“core”linkerpredatingthedivergenceofthefourgenes.Assumingthecoreismandatoryifanyotherlinkerexonsaretobesplicedin,weobservedalargefractionofallowablelinkersequencesforeachgene.CaMKIIβvariantsincorporatingproline-richexons19,20,or21andCaMKIIγvariantsincorporatingF-actin-interactingexon13,whichwerenotdetectedinourexperiment,aretwonotableexceptions.ThevariablelinkeraffectsactivationofCaMKIIα,butnotCaMKIIβWefocusedontheactivationpropertiesofCaMKIIαandβvariantsbecauseoftheirwell-documentedinvolvementinlearningandmemory(9,11).First,wedeterminedtheEC50valuesforCa2+/CaMactivationofallthreeCaMKIIαvariantssequencedfromhumanhippocampus.ForallEC50measurements,thecurvefitisshowninFigure3forsimplicity,andfitswithalldata


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pointsusedareshowninSupplement3.Forbrevitywerefertovariantsbylinkerlength:α-41(α14,15,18inFig.2C),α-30(α14,18),andα-0(α-no-linker).Inagreementwithpreviousexperimentscomparingα-30toα-0(19),wealsoobservedasignificantright-shiftintheEC50value(from24nMto313nM,respectively,Fig.3A).CaMKIIα-41(EC50=25nM,Fig.3A)didnotshowasignificantdifferencecomparedtoα-30.Next,weexaminedCaMKIIβvariantswiththebroadestpossiblerangeofvariablelinkerlengths:previouslyannotatedanddetectedvariantsβ-93,β-69,β-68,β-54,β-30,andβ-0,aswellastheannotated,butundetected,variantβ-217(Figure3B).Quitesurprisingly,weobservednosignificantdifferencesinEC50acrosstheentirerangeoflinkerlengths,withEC50ofallβvariantsfallinginthenarrowrangebetween14-31nM.ThisresultshowsthatthevariablelinkerdoesnottuneactivationthesamewayindifferentCaMKIIgenes.Thehillcoefficients(nH)fromthesemeasurementsofCaMKIIαandβvariantswereallquitesimilar,rangingfrom1.5to2.5,whichisconsistentwithpreviousmeasurements(19).Ofnote,thecooperativityinCaMKIIβvariantsisconsistentlyhighercomparedtoCaMKIIα,butdoesnotcorrelatewithEC50valuedifferences.Weobserveda15-folddifferenceintheEC50forCa2+/CaMbetweenα-0andβ-0(313nMvs21nM).Thedifferenceinactivationpropertiesofthesetwovariantsmustbedeterminedbysequencedifferencesoutsideofthevariablelinker:inthekinasedomain,thehubdomain,oracombinationofthetwo.Infact,theonlyCaMKIIβvariantwetestedwithanEC50outsideofthenarrowrangearound20nMwasthenovelvariantβ-93-short-hub,whichisslightlyright-shiftedfromtheotherCaMKIIβvariants(Fig.3B).Theonlydifferencebetweenβ-93(25nMEC50)andβ-93-short-hub(43nMEC50)are26residuesmissinginthehubdomain,indicatingthathubalonemayaffectactivationproperties.Theseresultsmotivatedournextexperiments.ThehubisdominanttothekinaseandvariablelinkerindeterminingactivationpropertiesWedirectlyassessedtheregulatorycontributionofthekinaseandhubdomainsofCaMKIIαandβ(90%and77%identity,respectively).Todothis,wecreatedtwochimeraswith0aalinkers.ThefirstchimeraistheCaMKIIαkinasedomainfusedtotheCaMKIIβhubdomain,andthesecondchimeraistheCaMKIIβkinasedomainfusedtotheCaMKIIαhubdomain.TheEC50valueoftheβkinase/αhubchimerawassignificantlyright-shiftedcomparedtothatoftheαkinase/βhubchimera(Fig.3C).BothCaMKIIβ-0andthechimerawiththeβhubhaveroughlythesameEC50(24nMand21nM,respectively).Ontheotherhand,CaMKIIα-0andthechimerawiththeαhubarebothright-shifted.However,theEC50ofthechimeraisstill3-foldlowerthantheEC50ofwildtypeCaMKIIα-0:102nMvs313nM.Thissuggeststhattheroleofthehubdomainindeterminingactivationpropertiesisdominantwithrespecttothekinasedomain,withnocontributionfromthekinaseinthecontextofβhub,butasignificantcontributioninthecontextofαhub.ActivationpropertiestrackwithevolutionarydivergenceWecomparedthedivergenceofhumanCaMKIIkinaseandhubdomainsusingthecorrespondingdomainsfromthesingleCaMKIIgeneinflyasoutgrouptorootthetrees(Fig.


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1D).Thekinaseandhubtreeshaveconsistenttopologies,inwhichthecommonancestorofCaMKIIαandδdivergedfromthecommonancestorofβandγ.However,whilethefourkinasedomainsdivergedinnearlyastartopology(thesegmentsseparatingtheα/δandβ/γancestorsfromtheircommonancestoraremuchshorterthananyothersegment),hubdivergencewasnotnearlyasuniform.CaMKIIαandδaremuchmoredivergedfromeachotherandfromβ/γ.CaMKIIβandγhubshavedivergedbyfartheleast,withthefewestsubstitutionsbetweenthem.Basedonthissimilarity,andtheapparentinvolvementofthehubdomaininactivation,wehypothesizedthatγ-0wouldbehavesimilarlytoβ-0.Wemeasuredactivationofγ-0andδ-0(Fig.3C),confirmingourhypothesis(γ-0:EC50=28nM).Consistentwiththeorderinwhichα,β/γ,andδsplit,aswellaswiththegreaterdegreeofdivergencebetweenαandδhubs,δ-0requiredanintermediateamountofCa2+/CaMforactivation(EC50=167nM):roughly7-foldhigherthanβ-0,but2-foldlowerthanα-0.DISCUSSIONForthefirsttime,weunambiguouslydeterminedatleast56CaMKIItranscriptspresentinahumanhippocampalsampleusingIlluminasequencing.Usingthesesequences,andotherspreviouslyannotatedfromvarioustissues,weaskedwhatarethedifferencesinregulationbetweenCaMKIIgenesandsplicevariants.WediscoveredthatwhileCaMKIIαregulationisdependentonthevariablelinkerregion,CaMKIIβisnot.Further,wenowshowanovelroleforthehubdomainintheregulationofCaMKIIactivationbyCa2+/CaM.OurdataprovideimportantinsightsintotheheterogeneityofCaMKIItranscriptsinahumantissueandalsoimplicateanewroleforthehubdomaininregulatingCaMKIIactivitythroughanallostericmechanism.ThisfindingopensanewwindowtoallostericcontrolofCaMKIIactivitythroughmodulationofthehubdomain.WesequencedasignificantnumberoftranscriptsfromallfourCaMKIIgenes.Notably,CaMKIIαproducedano-linkertranscript(CaMKIIα-0)thataccountedforover3%ofmappedreads.Thissequencewaspreviouslycrystallizedtoobtaintheonlyfull-lengthatomicresolutionstructureofCaMKII.Ourdatademonstratethepotentialphysiologicalrelevanceofthecompactconformationobservedinthatstructure(19).Thesheernumberofdetectedvariantsinhippocampalneurons(56acrossthefourCaMKIIgenes)suggeststhateithersplicingisnottightlyregulatedateveryexon,or,conversely,therearecomplexmechanismsofregulation.Intermsofabundanceoftranscriptswedetected,PCRbiasisunlikelytobesolelyresponsiblefortheroughly1000-folddifferencesbetweenthemost-andleast-frequentlyobservedvariantsofCaMKIIβ,γ,andδ.Thissuggeststhatsomevariantsaredeliberatelyup-regulatedrelativetoothers.Furthermore,wedidnotdetectevenasinglereadmappingtosomeofthepreviouslyannotatedvariants(forinstanceCaMKIIβvariantsincorporatingexon19,20,or21,andCaMKIIγvariantscontainingexon13).Thissuggeststhatsomevariantsarespecificallydown-regulated.WefoundthatagarosegelseparationoftranscriptsfollowedbySangersequencingofindividualbandscannotresolveCaMKIIvariantsfromhippocampus(Supplement4).Thisis


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becausesequencingtracesfrommostbandsclearlyindicatedamixtureoftemplatespresentinthesequencedsample.Inmanycases,carefulanalysisoftheSangersequencingtraceallowedustoidentifyatleasttwodistinctsplicevariants.Therefore,itisdifficulttointerpretthepresenceorabsenceofanexpectedgelbandasindicativeofthepresenceorabsenceofaparticularCaMKIItranscriptinasample.SinceeachIlluminareadobtainedbyourapproachreflectsasingletemplatemolecule,itproducesasignificantlymorerobustsurveyoftheCaMKIItranscripts.WesuggestthatthisapproachshouldbeappliedtoothertissuestobegintocatalogthediversityinCaMKIItranscripts.Weexpectthatinothertissuestheidentityandrelativeabundanceoftranscriptswillvaryfromthoseweobservedinthehippocampus.Withthisinformationinhand,wewillcertainlyimproveourunderstandingoftheregulationofalternativesplicing,andultimatelybeabletolinkthisregulationtothebiologicalneedswithineachtissue.Finally,ourresultscomparingtheEC50valuesbetweenCaMKIIαandβsplicevariantsshowthattheregulationofCaMKIIactivationisgene-dependentandsignificantlyaffectedbythesequenceofhubdomain.ThevariablelinkerinCaMKIIαdoesplayasignificantroleinregulatingCaMKIIαactivity,whichisconsistentwithpreviouswork(19).WeusedcartoonholoenzymesinFigure3toillustratethecompactandextendedautoinhibitedstatesofCaMKII,whichcorrespondtolowerandhighersensitivitiestoCa2+/CaM.ForCaMKIIα,thesecartoonsarebasedonacrystalstructure(PDB:3SOA)andSAXSmeasurements,respectively(19).Surprisingly,thevariablelinkerinCaMKIIβdoesnotplayasignificantroleinregulatingCaMKIIβactivity.UsingtheseEC50measurements,wehavedescribedCaMKIIactivationatfixedCa2+/CaMconcentrations.CaMKIIactivityisalsodependentonthefrequencyofCa2+delivered(19,30,36).Futureexperimentswillbeneededtocarefullydissectthiskineticcontributionindifferentsplicevariants.OurresultswithCaMKIIαandβchimerasprovideevidencethatthehubistheprimarydeterminantofsensitivitytoCa2+/CaM.AnalysisoftheCaMKIIαholoenzymecrystalstructureshowsthatthesubstitutedresiduescomparingCaMKIIαandβhubdomainsaredistributedfairlyevenlythroughoutthedomain(Fig.4A).Noneofthesesubstitutionsarefoundattheinterfacebetweenthehubandregulatorysegment,whichwouldbecleartointerpret.TheextremeC-terminusdoesvaryslightlyfromgenetogene(SVLPHinCaMKIIα),whichmaybeimplicatedinoligomerizationeffects(Fig.4B).Takentogether,wehypothesizethatthiseffectofthehubonkinaseactivityisbasedondynamics;wherebreathingmotionsorstabilitywithinthehubdomainallostericallyregulatestheaccessibilityofthecalmodulin-bindingsegment.Furtherexperimentswillbeneededtofullyelucidatethiscomplexmechanism.MATERIALSANDMETHODSInitialamplificationoftranscriptvariableregionscDNAwasreverse-transcribedfromacommerciallypurchasedhumanhippocampalRNAsample(BioChain)usingtheProtoScriptIIfirststrandcDNAsynthesiskit(NEB#E6560)witholigodTpriming.VariableregionsoftranscriptswerePCR-amplifiedfor35cyclesusinghigh-specificityprimerpairsdesignedtominimizecross-hybridizationbetweenCaMKIIgenesand


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Phusionpolymerase(NEB#M0530).Thisyieldedamixedpopulationofvariable-lengthampliconsreflectingthesplicevariantsofthetargetedgenepresentintheRNAsample.ConstructionofIlluminaSequencingLibrariesIlluminasequencinglibrarieswerepreparedfromtheinitialvariable-lengthampliconpoolsusinganadaptedPCR-basedconstructionprotocol(37).Briefly:first,gene-specificprimerswithoverhangscontainingIllumina-specifiedsequences(Figure2B)wereusedtosub-amplifytranscriptvariableregions.Second,genericprimersannealingtotheoverhangsintroducedinthefirstreactionwereusedtoincorporateannealingsitesforIlluminasequencingprimers,NEBNext(NEB#E7335)libraryindices,andP5/P7flowcellannealingsequences.Theresultingampliconswereagarosegel-purifiedwithsizeselectionbasedonthetheoreticalminimalandmaximallengthsofthevariableregioninserts.AnalysisofIlluminaSequencingIlluminareadsweregroupedbysequencesimilarity,producingclusterswith>90%sequenceidentityandtwoorfewerbasepairinsertions/deletions.TheconsensussequenceofeachclusterwasalignedtoexonsequencesfromthecorrespondingCaMKIIgeneobtainedfromthereferencehumangenome(GRCh38.p12,Ensemblrelease95)inordertomapexonorderinthetranscriptrepresentedbythecluster.Over90%ofreadsfromeachIlluminalibrarycouldbeclusteredandunambiguouslymappedwithreferenceexons.Weconsiderthese“mappable”reads.Python2codeimplementingreadclusteringandexonmappingisavailableuponrequest.ExpressionplasmidconstructionFull-lengthCaMKIIvariantswereclonedintoapETvectorcontainingN-terminal6xHisfollowedbyaSUMOtag.CaMKIIchimeraswereconstructedasfollows.Thekinase/regulatorysegmentandhubdomainsofCaMKIIαandCaMKIIβwereamplifiedfromfull-lengthconstructs.Thekinasedomainincludestheregulatorysegmentofitscorrespondingkinase.Eachdomainwasamplifiedwitha20bpsoverhangthatwasthecorrespondingCaMKIIgene.ThechimeraswereassembledusingtheGibsonassemblytechnique.ProteinexpressionandpurificationAllCaMKIIvariantsusedforthisexperimentwererecombinantlyexpressedandpurifiedaccordingto(38).PlasmidsencodingCaMKIIvariantswereco-transformedwithlambdaphosphataseintoRosetta2(DE3)pLysScompetentcells(Millipore).Expressionwasinducedovernightwith1mMIsopropylβ-D-1-thiogalactopyranoside(IPTG)andculturesweregrownovernightat18°C.CellpelletswereresuspendedinBufferA(25mMTris-HClpH8.5,150mMKCl,50mMImidazole,10%glycerol)with25mMMagnesiumchloride,containingacocktailofproteaseinhibitorsandDNAse(AEBSF0.2mM,Leupeptin0.005mM,1ug/mLPepstatin,1ug/mLAprotinin,0.1mg/mLTrypsinInhibitor,0.5mMBenzamidine,1ug/mLDNAse)andlysed.AllsubsequentpurificationstepswereperformedusinganÄktapurechromatographysystemat4°C.Clarifiedlysatewasloadedontoa5mLHisTrapFFNiNTASepharosecolumn(GE),andelutedwithacombinationof50%BufferAand50%BufferB(25mMTris-HClpH8.5,


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150mMKCl,1Mimidazole,10%glycerol)forafinalconcentrationof0.5Mimidazole.TheproteinwasdesaltedofresidualimidazoleusingaHiPrep26/10Desaltingcolumn,andHisSUMOtagswerecleavedwithUlp1proteaseovernightat4°CinBufferC(25mMTris-HClpH8.5,150mMKCl,2mMTCEP,50mMImidazole,10%glycerol).CleavedtagswereseparatedbyasubtractiveNiNTAstep.Next,ananionexchangestepwasdoneusinga5mLHiTrapQ-FFandproteinwaselutedwithaKClgradient.Elutedproteinswereconcentratedandfurtherpurifiedingelfiltrationbuffer(25mMTris-HClpH8.0,150mMKCl,1mMTCEP,10%glycerol)usingaSuperose6Increase10/300sizeexclusioncolumn(GE).Purefractionswerethenconcentrated,aliquoted,flashfrozeninliquidnitrogenandstoredat-80°Cuntilfurtheruse.Calmodulin(Gallusgallus)wasrecombinantlyexpressedfromapET-15bvector(generousgiftfromAngusNairn)inBL21(DE3)cells(Millipore)andpurifiedaspreviouslydescribed(PutkeyandWaxham,1996).Toquantifythecalmodulinconcentrationformakingstockstouseinthekinaseassays,weusedcirculardichroismonaJascoJ-1500Spectrophotometertomakeameasurementintriplicateforourpurifiedsamplescanningawavelengthspectrumbetween250-215nmtomeasurethecharacteristicwavelengthof222nmaccordingto(39).Equationusedtocalculatecalmodulinconcentration:[CaM](mM)=(1000*(mdegat222nmsample-mdegat222nmblank))/(ϴ*ℓ*(#aminoacids))ℓ=pathlength,ϴ=molarellipticity

calmodulin nM = 1000 × (𝐶𝐷!"#$%&!!!!" − 𝐶𝐷!"#$%!!!!")Θ × 𝑙 × number of amino acids

wherecirculardichroismat222nm(𝐶𝐷!!!!")isexpressedinmdeg,Θisthemolarellipticity,and𝑙isthepathlengthincm.CoupledkinaseassaysKinaseactivitywasmonitoredwithpreviouslydescribedconditions(Barkeretal.1995;Chaoetal.2010)usingaSynergyH1microplatereader(Biotek).Theadditionofcalmodulin(concentrationsrangingfrom0nM-2µM)tothereactionwasusedtoinitiateCaMKIIactivity,afterwhichabsorbancewasmeasuredat15secondintervalsfor10minutes.Thechangeinabsorbanceoverthecourseoftheresultingtimeseriesofmeasurementswasfitwithastraightline(y=mx+c)toobtainaslope(m)proportionaltothekineticrateofthereaction.Foreachtimeseries,slopeswerefittoaslidingwindowof5points(1minute15seconds)andthemaximumobservedslopewasusedtorepresentthekineticrateofthatreaction.Kineticratesacrossaseriesofcalmodulinconcentrationswerefitwiththefollowingequation:

𝑌 = 𝑌!"# + 𝑌!"# − 𝑌!"# ∙ 𝑋!!𝑋!! + 𝐸𝐶!"!!

toobtainEC50(definedasthecalmodulinconcentrationneededtoreachhalfthemaximalreactionvelocity)andcooperativityvalues(Hillcoefficients,nH).95%confidenceintervalsforfitparameters(EC50andnH)weredeterminedusingthefollowingbootstrapprocedure.10,000


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replicatecalmodulinconcentrationseriesweregeneratedbyrandomlyselectingoneobservedkineticrateateachmeasuredcalmodulinconcentrationfromthesetofreplicatesforthatvariant.Eachgeneratedconcentrationserieswasfitwiththeequationabove.Parametervaluesatthe2.5thand97.5thquantilesofthe10,000fitsweretakenastheboundariesofthe95%confidenceinterval.AcknowledgementsWethankLukeChao,FlorianHeydandAndreasFranzforhelpfuldiscussionsondataandthemanuscript.WealsothankPeterChien,EricStrieter,ScottGarmanforhelpfuldiscussions.REFERENCES1. Lin,Y.C.,andRedmond,L.(2009)NeuronalCaMKIIactsasastructuralkinase.

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20. Ma,H.,Groth,R.D.,Cohen,S.M.,Emery,J.F.,Li,B.,Hoedt,E.,Zhang,G.,Neubert,T.A.,andTsien,R.W.(2014)gammaCaMKIIshuttlesCa(2)(+)/CaMtothenucleustotriggerCREBphosphorylationandgeneexpression.Cell159,281-294

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27. Bayer,K.U.,Lohler,J.,andHarbers,K.(1996)Analternative,nonkinaseproductofthebrain-specificallyexpressedCa2+/calmodulin-dependentkinaseIIalphaisoformgeneinskeletalmuscle.MolCellBiol16,29-36

28. Bayer,K.U.,Harbers,K.,andSchulman,H.(1998)alphaKAPisananchoringproteinforanovelCaMkinaseIIisoforminskeletalmuscle.EmboJournal17,5598-5605

29. Chao,L.H.,Pellicena,P.,Deindl,S.,Barclay,L.A.,Schulman,H.,andKuriyan,J.(2010)IntersubunitcaptureofregulatorysegmentsisacomponentofcooperativeCaMKIIactivation.NatStructMolBiol17,264-272

30. DeKoninck,P.,andSchulman,H.(1998)SensitivityofCaMkinaseIItothefrequencyofCa2+oscillations.Science279,227-230

31. Saitou,N.,andNei,M.(1987)Theneighbor-joiningmethod:anewmethodforreconstructingphylogenetictrees.MolBiolEvol4,406-425

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34. Chang,H.Y.,Minahan,K.,Merriman,J.A.,andJones,K.T.(2009)Calmodulin-dependentproteinkinasegamma3(CamKIIgamma3)mediatesthecellcycleresumptionofmetaphaseIIeggsinmouse.Development136,4077-4081

35. Mukherjee,S.,Sheng,W.,Sun,R.,andJanssen,L.J.(2017)Ca(2+)/calmodulin-dependentproteinkinaseIIbetaandIIdeltamediateTGFbeta-inducedtransductionoffibronectinandcollageninhumanpulmonaryfibroblasts.AmJPhysiolLungCellMolPhysiol312,L510-L519

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FOOTNOTES Funding was provided by the University of Massachusetts, Amherst and a grant to M.M.S. from the National Institute of General Medical Sciences (R01GM123157).


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MAINFIGURES

Figure1.CaMKIIsequencedivergenceandstructuralorganization.(A)EachsubunitofCaMKIIconsistsofakinasedomain,regulatorysegment,variablelinkerregion,andhubdomain.Theregulatorysegmenthousesthecalmodulinbindingregionaswellasaregulatoryautophosphorylationsite,Thr286.(B)Theregulatorysegmentactsasanautoinhibitorydomainbyblockingthesubstrate-bindingpocket.InthepresenceofCa2+,Ca2+-CaMbindstheregulatorysegment,exposingthesubstrate-bindingpocketandturningtheenzymeon.(C)ThehubdomainoligomerizesCaMKIIintotwostackedhexamericrings.ShownisacartoonofanactivestructurewithCa2+-CaMboundtotheregulatorysegment.(D)NeighborJoiningreconstructionsofkinaseandhubdomaindivergenceforthefourhumanCaMKIIgenes,rootedwithD.melanogasterCaMKIIoutgroups.


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Figure2.IlluminasequencingrevealsaheterogeneouspopulationofCaMKIIvariantsinthehumanhippocampus.(A)HumanCaMKIIgenesareencodedby24exonswithclearone-to-onecorrespondenceacrossgenes.Exons1-10encodethecatalytickinasedomain,11and12encodetheregulatorysegment,13-21encodethevariable-lengthlinker,and22-24encodetheassociation(hub)domain.Missinglinkerexonsindicatethattheexonisnotencodedinthecorrespondinggene.Constitutivelyincorporatedkinase,regulatorysegment,andhubexons(grey)aresplicedintoeverytranscript.CaMKIIδencodestwoversionsofkinaseexon6,withoneortheother,butnotboth,incorporatedintotranscripts.Severalexonscontainalternativesplicesites,permittingomissionofafractionoftheexon(indicatedbyhatching).CaMKIIβ


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encodesthreenearlyidenticalproline-richlinkerexons:19-21,homologoustoCaMKIIγexon19.(B)IlluminalibraryconstructionisillustratedwithCaMKIIβ.InitialPCRamplificationofaregionencompassingallalternativesplicesites(includingthelinkerandhubexon23)iscarriedoutwithprimersdesignedforspecificityfortargetCaMKIIgene,totheexclusionoftheotherthreegenes(orangeandredarrows).InitialampliconsserveastemplatesforamplificationoflibraryinsertsinasecondPCRreactionwithadifferentprimerpair(greenandbluearrows).Resultinginsertsarefullyspannedbypaired-end251bpreadsfornearlyallsplicevariants.ForCaMKIIβ,thisistrueaslongasexons19,20,and21arenotincorporated.Theseexonsareshownassemi-transparentbecausetheywerenotincorporatedinanydetectedhippocampalsplicevariant.(C)SummaryofCaMKIIαandβtranscriptvariantsdetectedbyIlluminasequencing.Alldetectedvariants,includingallpreviouslyannotatedvariants,areshownforCaMKIIα.ForCaMKIIβ,thetopeightread-mappingvariantsareshownabovetheblacklineandtheremainingpreviouslyannotatedvariantsareshownbelowtheline.Variantsareconsiderednoveliftheyhavenotbeenpreviouslyannotated.Variantshighlightedinbluearethosethatwereusedinactivitymeasurements(seeFigure3).


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Figure3.ThehubdomainplaysacrucialroleinCa2+/CaMsensitivity.CaMKIIactivityagainstapeptidesubstrate(syntide)wasmeasuredasafunctionofCa2+-CaMconcentration.Tosimplifycomparisons,fitcurveswerenormalizedbythecorrespondingVmaxfitparameteranddatapointsarenotshown(seeSupplement3).TheEC50valueforeachfitisindicatedbyasinglebox,anddashedgraylinesinB-DindicatereferenceEC50valuesforCaMKIIα-30andCaMKIIα-0(B)orCaMKIIβ-0andCaMKIIα-0(C,D),aslabeledontheX-axis.ActivitymeasurementsandEC50andnH(Hillcoefficient)fitparametersareshownforallthreeCaMKIIαsplicevariants(A),eightselectedCaMKIIβvariants(B),chimerasofCaMKIIα/βwithnolinker(C),andCaMKIIγandδ


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withnolinker(D).EC50andHillcoefficient(nH)fitparameterswith95%confidenceintervals(seeMaterialsandMethods)arelistedforeachtestedvariant.CartoonrepresentationsofCaMKIIholoenzymesintheirautoinhibitedstatesareshowninpanelA.ForpanelsCandD,eachdomainislabeledaccordingtotheCaMKIIgene.

Figure4.AllostericcontributionsfromthehubdomainregulateCaMKIIactivation.(A)The32positionsthatdifferbetweenCaMKIIαandβhubsarerepresentedbyspheresatthealphaCarbonpositiononthestructureofCaMKIIαhub(3SOA).Hubisshownincyan,regulatorysegmentinorange,andkinasedomainingreen.(B)SequencealignmentofCaMKIIα,β,γ,andδhubdomains,coloredbydegreeofconservation(darktolight=mosttoleastconserved).Forconvenience,allhubsequencesstartatresidue1.ResiduesinCaMKIIαhubthatdifferinCaMKIIβarehighlightedbyredboxes.


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SUPPLEMENTALINFORMATIONSupplement1:IlluminasequencingresultsforallCaMKIIgenes.

Variantname Ensembl identifierCCDS

identifier

NCBI

NameNCBI identifier

Read

count

%of

reads

CaMKIIαα14,18 ENST00000348628 CCDS43386 var.2 NM_171825 183118 94.6

α-no-linker novel 7527 3.89

α14,15,18 ENST00000398376 CCDS43387 var.1 NM_015981 2941 1.52

Variantname Ensembl identifier CCDSidentifier

NCBIName NCBI identifier Read

count%ofreads

CaMKIIββ13,14b,16,17,18 ENST00000350811 CCDS5484 var.2 NM_172078 65577 44.8

β14a,16,18 ENST00000358707 CCDS5486 var.5 NM_172081 20153 13.8

β14a,16,17,18 ENST00000395747 CCDS5485 var.3 NM_172079 13327 9.11

β14b,16,17,18 ENST00000258682 CCDS43573 var.4 NM_172080 10362 7.08

β14b,16,18 novel var.X20 XM_017012664 9192 6.28

β13,14b,16,18 novel var.X24 XM_017012667 8616 5.89

β13,14b,17,18 novel var.X26 XM_017012669 5805 3.97

β13,14b,16,17,18-shorthub novel 5442 3.72

β14b,17,18 novel var.X22 XM_017012665 1700 1.16

β14a,17,18 novel var.X21 XM_005249864 1218 0.83

β13,14b,18 novel 928 0.63

β14a,18 ENST00000353625 CCDS5488 var.7 NM_172083 750 0.51

β14b,16,17,18-shorthub novel 634 0.43

β13,14a,16,17,18 novel 412 0.28

β13,14b,16,18-shorthub novel 400 0.27

β14b,18 novel 352 0.24

β14a,16,17,18-shorthub novel 309 0.21

β14a,16,18-short hub novel 303 0.21

β-no-linker ENST00000346990 CCDS5489 var.8 NM_172084 185 0.13

β13,14a,16,18 novel 169 0.12

β14a,17,18-short hub novel 155 0.11

β13,14b,17,18-shorthub ENST00000347193 CCDS5487 var.6 NM_172082 148 0.10

β14b,16,18-short hub novel 104 0.071

β13 novel 52 0.036


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Variantname Ensembl identifierCCDS

identifier

NCBI

NameNCBI identifier

Read

count

%of

reads

CaMKIIδδ61,14b,18 ENST00000514328 CCDS82950 var.15 NM_001321574 45214 53.0

δ61,14a,18

ENST00000342666

ENST00000379773

ENST00000394524

CCDS3703

CCDS3704

CCDS43263

var.3

var.4

var. 2

NM_001221

NM_172127

NM_172128

24712 29.0

δ61,14a,16,17,18 novel 6483 7.64

δ62,14b,18 novel 4001 4.71

δ62,14a,18 novel 1225 1.44

δ61,14a,17,18 ENST00000394522 CCDS47127 var.6 NM_172114 1121 1.32

δ61,14b,17,18 novel 820 0.97

δ62,14a,17,18 novel 563 0.66

δ61,14b novel 431 0.51

δ61,14a novel 326 0.38

δ61,14b,16,17,18 novel 84 0.10

δ61,14a,15,17,18 novel 90 0.11

VariantnameEnsembl

identifier

CCDS

identifier

NCBI

NameNCBI identifier

Read

count

%of

reads

CaMKIIγγ14,16a,17,18b,19a ENST00000322680 CCDS7338 var.1 NM_172171 53510 31.3

γ14,16a,17,18b ENST00000322635 CCDS7337 var.2 NM_172169 47800 28.0

γ14,15,17,18b var.12 NM_001367519 19801 11.6

γ14,17,18b,19a var.21 NM_001367528 14773 8.65

γ14,15,17,18b,19a var.11 NM_001367518 12127 7.09

γ14,17,18b ENST00000351293 CCDS7336 var.4 NM_001222 10268 6.01

γ14,16b,17,18b,19a var.13 NM_001367520 4586 2.68

γ14,16b,17,18b var.6 NM_172173 3253 1.90

γ14,15,16a,17,18b ENST00000305762 var.24 NM_001367531 2196 1.29

γ14,15,16a,17,18b,19a var.40 NM_001367547 1662 0.97

γ14,18b,19a novel 160 0.094

γ14,15,18b novel 133 0.078

γ14,15 novel,premature termination 111 0.065

γ14,15,16b,17,18b novel var.X32 XM_017016730 100 0.059

γ14,17,18a,19b novel 96 0.056

γ14,18b novel 81 0.047

γ14,15,16b,17,18b,19a novel 72 0.042


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Supplement2:ClusterconsensussequencesforallCaMKIIgenes.CaMKIIalpha_proteinsequences>alpha14,18WISHRSTVASCMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCD>alpha-no-linkerWISHRSTVASCMHRQETVDCLKKFNARRKLKGAILTVMLATRNFSVRKQEIIKVTEQLIEAISNGDFESYTKMCD>alpha14,15,18WISHRSTVASCMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSGGKSGGNKKSDGVKKRKSSSSVQLMESSESTNTTIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCDCaMKIIalpha_DNAsequences>alpha14,18TGGATCTCGCACCGCTCCACCGTGGCATCCTGCATGCACAGACAGGAGACCGTGGACTGCCTGAAGAAGTTCAATGCCAGGAGGAAACTGAAGGGAGCCATTCTCACCACGATGCTGGCCACCAGGAACTTCTCCGGAGGGAAGAGTGGGGGAAACAAGAAGAGCGATGGTGTGAAGGAATCCTCAGAGAGCACCAACACCACCATCGAGGATGAAGACACCAAAGTGCGGAAACAGGAAATTATAAAAGTGACAGAGCAGCTGATTGAAGCCATAAGCAATGGAGATTTTGAGTCCTACACGAAGATGTGCGAC>alpha-no-linkerTGGATCTCGCACCGCTCCACCGTGGCATCCTGCATGCACAGACAGGAGACCGTGGACTGCCTGAAGAAGTTCAATGCCAGGAGGAAACTGAAGGGAGCCATTCTCACCGTGATGCTGGCCACCAGGAACTTCTCCGTGCGGAAACAGGAAATTATAAAAGTGACAGAGCAGCTGATTGAAGCCATAAGCAATGGAGATTTTGAGTCCTACACGAAGATGTGCGAC>alpha14,15,18TGGATCTCGCACCGCTCCACCGTGGCATCCTGCATGCACAGACAGGAGACCGTGGACTGCCTGAAGAAGTTCAATGCCAGGAGGAAACTGAAGGGAGCCATTCTCACCACGATGCTGGCCACCAGGAACTTCTCCGGAGGGAAGAGTGGGGGAAACAAGAAGAGCGATGGTGTGAAGAAAAGAAAGTCCAGTTCCAGCGTTCAGTTAATGGAATCCTCAGAGAGCACCAACACCACCATCGAGGATGAAGACACCAAAGTGCGGAAACAGGAAATTATAAAAGTGACAGAGCAGCTGATTGAAGCCATAAGCAATGGAGATTTTGAGTCCTACACGAAGATGTGCGAC


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CaMKIIbeta_proteinsequences>beta13,14b,16,17,18ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14a,16,18ILTTMLATRNFSAAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14a,16,17,18ILTTMLATRNFSAAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14b,16,17,18ILTTMLATRNFSAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14b,16,18ILTTMLATRNFSAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta13,14b,16,18ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta13-14b-17-18ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta13,14b,16,17,18-short-hubILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta14b,17,18ILTTMLATRNFSAKSLLNKKADGVKEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14a-17-18ILTTMLATRNFSAAKSLLNKKADGVKEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta13,14b,18


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ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14a,18ILTTMLATRNFSAANSLLNKKADGVKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14b,16,17,18ILTTMLATRNFSAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta13,14b,16,18ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta14b,18ILTTMLATRNFSAKSLLNKKADGVKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14a,16,17,18ILTTMLATRNFSAANSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta14a,16,18ILTTMLATRNFSAAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta13,14a,16,17,18ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta-no-linkerILTTMLATRNFSARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta13,14a,16,18ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAAKSLLNKKADGVKPQTNSTNNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLL>beta14a,17,18-short-hubILTTMLATRNFSAAKSLLNKKADGVKEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta13,14b,17,18-short-hub


https://doi.org/10.1101/721589

23

ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQAKSLLNKKADGVKEPQTTVIHNPVDGIKESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta14b,16,18-short-hubILTTMLATRNFSAKSLLNKKADGVKPQTNSTKNSAAATSPKGTLPPAALESSDSANTTIEDEDAKARKQEIIKTTEQLIEAVNNGDFEAYAFYFENLL>beta13ILTTMLATRNFSVGRQTTAPATMSTAASGTTMGLVEQARKQEIIKTTEQLIEAVNNGDFEAYAKICDPGLTSFEPEALGNLVEGMDFHRFYFENLLCaMKIIbeta_DNAsequences>beta13,14b,16,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14a,16,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14a,16,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGC


https://doi.org/10.1101/721589

24

TAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14b,16,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14b,16,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta13,14b,16,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta13-14b-17-18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGCCTCAA


https://doi.org/10.1101/721589

25

ACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta13,14b,16,17,18-short-hubCATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta14b,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14a-17-18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta13,14b,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGTCTTCT


https://doi.org/10.1101/721589

26

GACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14a,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAATAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14b,16,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta13,14b,16,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta14b,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14a,16,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAATAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGC


https://doi.org/10.1101/721589

27

CCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta14a,16,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta13,14a,16,17,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta-no-linkerCATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta13,14a,16,18CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAACAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGG


https://doi.org/10.1101/721589

28

CCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTG>beta14a,17,18-short-hubCATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta13,14b,17,18-short-hubCATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGGAGCCTCAAACCACCGTCATCCATAACCCAGTGGACGGGATTAAGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta14b,16,18-short-hubCATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGCCAAGAGTTTACTCAACAAGAAAGCAGATGGAGTCAAGCCCCAGACGAATAGCACCAAAAACAGTGCAGCCGCCACCAGCCCCAAAGGGACGCTTCCTCCTGCCGCCCTGGAGTCTTCTGACAGTGCCAATACCACCATAGAGGATGAAGACGCTAAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCATTCTACTTCGAGAACCTGCTG>beta13CATCCTCACCACCATGCTGGCCACACGGAATTTCTCAGTGGGCAGACAGACCACCGCTCCGGCCACAATGTCCACCGCGGCCTCCGGCACCACCATGGGGCTGGTGGAACAAGCCCGGAAGCAGGAGATCATTAAGACCACGGAGCAGCTCATCGAGGCCGTCAACAACGGTGACTTTGAGGCCTACGCGAAAATCTGTGACCCAGGGCTGACCTCGTTTGAGCCTGAAGCACTGGGCAACCTGGTTGAAGGGATGGACTTCCACAGATTCTACTTCGAGAACCTGCTGCaMKIIdelta_proteinsequences>Fread,delta6v1,14b-18SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14b-18


https://doi.org/10.1101/721589

29

ICQRSTVASMMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSAKSLLKKPDGVKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v1,14a-18SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14a-18CQRSTVASMMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSAAKSLLKKPDGVKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v1,14a-16-17-18SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14a-16-17-18TMLATRNFSAAKSLLKKPDGVKINNKANVVTSPKENIPTPALEPQTTVIHNPDGNKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v2,14b-18SEADASHCIQQILEAVLHCHQMGVVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v2,14b-18ICQRSTVASMMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSAKSLLKKPDGVKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v2,14a-18SEADASHCIQQILEAVLHCHQMGVVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLLKDPY>Rread,delta6v2,14a-18CQRSTVASMMHRQETVDCLKKFNARRKLKGAILTTMLATRNFSAAKSLLKKPDGVKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v1,14a-17-18SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14a-17-18TVDCLKKFNARRKLKGAILTTMLATRNFSAAKSLLKKPDGVKEPQTTVIHNPDGNKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v1,14b-17-18


https://doi.org/10.1101/721589

30

SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14b-17-18ETVDCLKKFNARRKLKGAILTTMLATRNFSAKSLLKKPDGVKEPQTTVIHNPDGNKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v2,14a-17-18SEADASHCIQQILEAVLHCHQMGVVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v2,14a-17-18TVDCLKKFNARRKLKGAILTTMLATRNFSAAKSLLKKPDGVKEPQTTVIHNPDGNKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v1,14bSEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14bQPCQTHHSLRGTEAPMDLSTFYCCFHDAQTGDCRLLEEI*C*KKTKGCHLDNYAGYKEFLSQEFVEETRWSKARKQEIIKVTE>Fread,delta6v1,14aSEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14aPCQTHHSLRGTEAPMDLSTFYCCFHDAQTGDCRLLEEI*C*KKTKGCHLDNYAGYKEFLSSQEFVEETRWSKARKQEIIKVTE>Fread,delta6v1,14b-16-17-18SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14b-16-17-18TTMLATRNFSAKSLLKKPDGVKINNKANVVTSPKENIPTPALEPQTTVIHNPDGNKESTESSNTTIEDEDVKARKQEIIKVTE>Fread,delta6v1,14a-15-17-18SEADASHCIQQILESVNHCHLNGIVHRDLKPENLLLASKSKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKDPY>Rread,delta6v1,14a-15-17-18


https://doi.org/10.1101/721589

31

RKLKGAILTTMLATRNFSAAKSLLKKPDGVKKRKSSSSVQMMEPQTTVIHNPDGNKESTESSNTTIEDEDVKARKQEIIKVTECaMKIIdelta_DNAsequences>Fread,delta6v1,14b-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14b-18GATCTGTCAACGTTCTACTGTTGCTTCCATGATGCACAGACAGGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14a-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14a-18CTGTCAACGTTCTACTGTTGCTTCCATGATGCACAGACAGGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14a-16-17-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAA


https://doi.org/10.1101/721589

32

AGATCCTTATG>Rread,delta6v1,14a-16-17-18AACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGATAAACAACAAAGCCAACGTGGTAACCAGCCCCAAAGAAAATATTCCTACCCCAGCGCTGGAGCCCCAAACTACTGTAATCCACAACCCTGATGGAAACAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v2,14b-18CAGTGAAGCTGATGCCAGTCATTGCATTCAGCAGATCCTGGAGGCTGTGCTACACTGCCATCAGATGGGCGTGGTCCATCGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v2,14b-18GATCTGTCAACGTTCTACTGTTGCTTCCATGATGCACAGACAGGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v2,14a-18CAGTGAAGCTGATGCCAGTCATTGCATTCAGCAGATCCTGGAGGCTGTGCTACACTGCCATCAGATGGGCGTGGTCCATCGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACTTAAAGATCCTTATG>Rread,delta6v2,14a-18CTGTCAACGTTCTACTGTTGCTTCCATGATGCACAGACAGGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14a-17-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACA


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33

GGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14a-17-18GACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGCCCCAAACTACTGTAATCCACAACCCTGATGGAAACAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14b-17-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14b-17-18GGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGCCCCAAACTACTGTAATCCACAACCCTGATGGAAACAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v2,14a-17-18CAGTGAAGCTGATGCCAGTCATTGCATTCAGCAGATCCTGGAGGCTGTGCTACACTGCCATCAGATGGGCGTGGTCCATCGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v2,14a-17-18GACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGGAGCCCCAAACTACTGTAATCCACAACCCTGATGGAAACAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAA


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AGTCACTGAAC>Fread,delta6v1,14bCAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14bTCAACCCTGCCAAACGCATCACAGCCTCAGAGGCACTGAAGCACCCATGGATCTGTCAACGTTCTACTGTTGCTTCCATGATGCACAGACAGGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14aCAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14aACCCTGCCAAACGCATCACAGCCTCAGAGGCACTGAAGCACCCATGGATCTGTCAACGTTCTACTGTTGCTTCCATGATGCACAGACAGGAGACTGTAGACTGCTTGAAGAAATTTAATGCTAGAAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14b-16-17-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14b-16-17-18GACAACTATGCTGGCTACAAGGAATTTCTCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGATAAACAACAAAG


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35

CCAACGTGGTAACCAGCCCCAAAGAAAATATTCCTACCCCAGCGCTGGAGCCCCAAACTACTGTAATCCACAACCCTGATGGAAACAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAAC>Fread,delta6v1,14a-15-17-18CAGTGAAGCTGATGCCAGTCATTGTATACAGCAGATTCTAGAAAGTGTTAATCATTGTCACCTAAATGGCATAGTTCACAGGGACCTGAAGCCTGAGAATTTGCTTTTAGCTAGCAAATCCAAGGGAGCAGCTGTGAAATTGGCAGACTTTGGCTTAGCCATAGAAGTTCAAGGGGACCAGCAGGCGTGGTTTGGTTTTGCTGGCACACCTGGATATCTTTCTCCAGAAGTTTTACGTAAAGATCCTTATG>Rread,delta6v1,14a-15-17-18AAGAAAACTAAAGGGTGCCATCTTGACAACTATGCTGGCTACAAGGAATTTCTCAGCAGCCAAGAGTTTGTTGAAGAAACCAGATGGAGTAAAGAAAAGGAAGTCCAGTTCGAGTGTTCAGATGATGGAGCCCCAAACTACTGTAATCCACAACCCTGATGGAAACAAGGAGTCAACTGAGAGTTCAAATACAACAATTGAGGATGAAGATGTGAAAGCACGAAAGCAAGAGATTATCAAAGTCACTGAACCaMKIIgamma_proteinsequences>gamma14,16a,17,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKPQSNNKNSLVSPAQEPAPLQTAMEPQTTVVHNATDGIKGSTESCNTTTEDEDLKAAPLRTGNGSSVPEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,16a,17,18bLTTMLVSRNFSAAKSLLNKKSDGGVKPQSNNKNSLVSPAQEPAPLQTAMEPQTTVVHNATDGIKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15,17,18bLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMEPQTTVVHNATDGIKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,17,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKEPQTTVVHNATDGIKGSTESCNTTTEDEDLKAAPLRTGNGSSVPEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15,17,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMEPQTTVVHNATDGIKGSTESCNTTTEDEDLKAAPLRTGNGSSV


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PEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,17,18bLTTMLVSRNFSAAKSLLNKKSDGGVKEPQTTVVHNATDGIKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,16b,17,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKPQSNNKNSLEPQTTVVHNATDGIKGSTESCNTTTEDEDLKAAPLRTGNGSSVPEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,16b,17,18bLTTMLVSRNFSAAKSLLNKKSDGGVKPQSNNKNSLEPQTTVVHNATDGIKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15,16a,17,18bLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMPQSNNKNSLVSPAQEPAPLQTAMEPQTTVVHNATDGIKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15,16a,17,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMPQSNNKNSLVSPAQEPAPLQTAMEPQTTVVHNATDGIKGSTESCNTTTEDEDLKAAPLRTGNGSSVPEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKGSTESCNTTTEDEDLKAAPLRTGNGSSVPEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15,18bLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15LTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMCENRRSLRLQNS*LKPSTMGTLRPTRRFVIQASLPLSLRPLVTSWR>gamma14,15,16b,17,18bLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMPQSNNKNSLEPQTTVVHNATDGIKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,17,18a,19bLTTMLVSRNFSAAKSLLNKKSDGGVKEPQTTVVHNATDGIKGSTESCNTTTEDEDLKARCLKDGAPGTEQPPLQACSPSLLSAPQPCENRRSLRLQNS*LKPSTMGTLRPTRRFVIQASLPLSLRPLVTSWR>gamma14,18b


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37

LTTMLVSRNFSAAKSLLNKKSDGGVKGSTESCNTTTEDEDLKVRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVE>gamma14,15,16b,17,18b,19aLTTMLVSRNFSAAKSLLNKKSDGGVKKRKSSSSVHLMPQSNNKNSLEPQTTVVHNATDGIKGSTESCNTTTEDEDLKAAPLRTGNGSSVPEGRSSRDRTAPSAGMQPQPSLCSSAMRKQEIIKITEQLIEAINNGDFEAYTKICDPGLTSFEPEALGNLVECaMKIIgamma_DNAsequences>gamma14,16a,17,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGCCACAGAGCAACAACAAAAACAGTCTCGTAAGCCCAGCCCAAGAGCCCGCGCCCTTGCAGACGGCCATGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,16a,17,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGCCACAGAGCAACAACAAAAACAGTCTCGTAAGCCCAGCCCAAGAGCCCGCGCCCTTGCAGACGGCCATGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,17,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTG


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AGGCCCTTGGTAACCTCGTGGAGGG>gamma14,17,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,17,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,17,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,16b,17,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGCCACAGAGCAACAACAAAAACAGTCTCGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAA


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AACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,16b,17,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGCCACAGAGCAACAACAAAAACAGTCTCGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,16a,17,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGCCACAGAGCAACAACAAAAACAGTCTCGTAAGCCCAGCCCAAGAGCCCGCGCCCTTGCAGACGGCCATGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,16a,17,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGCCACAGAGCAACAACAAAAACAGTCTCGTAAGCCCAGCCCAAGAGCCCGCGCCCTTGCAGACGGCCATGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCG


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GTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15CCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,16b,17,18bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGCCACAGAGCAACAACAAAAACAGTCTCGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,17,18a,19bCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,18b


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CCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGTGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG>gamma14,15,16b,17,18b,19aCCTCACGACCATGCTTGTCTCCAGGAACTTCTCAGCTGCCAAAAGCCTATTGAACAAGAAGTCGGATGGCGGTGTCAAGAAAAGGAAGTCGAGTTCCAGCGTGCACCTAATGCCACAGAGCAACAACAAAAACAGTCTCGAGCCACAAACCACTGTGGTACACAACGCTACAGATGGGATCAAGGGCTCCACAGAGAGCTGCAACACCACCACAGAAGATGAGGACCTCAAAGCTGCCCCGCTCCGCACTGGGAATGGCAGCTCGGTGCCTGAAGGACGGAGCTCCCGGGACAGAACAGCCCCCTCTGCAGGCATGCAGCCCCAGCCTTCTCTCTGCTCCTCAGCCATGCGAAAACAGGAGATCATTAAGATTACAGAACAGCTGATTGAAGCCATCAACAATGGGGACTTTGAGGCCTACACGAAGATTTGTGATCCAGGCCTCACTTCCTTTGAGCCTGAGGCCCTTGGTAACCTCGTGGAGGG


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Supplement3:CaMKIIactivitymeasurementswithalldatapointsshown.Individualdatasetsarecolor-coded.


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Supplement4:PCRampliconsfrommultipleCaMKIIβ variantsdonotseparateonagarosegel.APCRreactionwithCaMKIIβ-specificprimerswasloadedontoa2.5%agarosegelandrunat10Vforroughly24hrs(picturedbelow)foroptimalbandseparation.Thesinglevisiblebandwasexcised,purified,andSanger-sequenced.Afragmentoftheresultingsequencingtraceisshown,withheightofgreybackgroundrepresentingPhredqualityscores.Thefirst15bpofthefragment(leftofgreyline)maptothelast15bpofCaMKIIβconstitutiveexon12,incorporatedintoeveryCaMKIIβtranscript.Thelast15bpofthefragment(rightofgreyline)showadramaticincreaseofsecondarypeakheights,withacorrespondingdrop-offinPhredscores.Thesequencedefinedbyprimarypeaks(TGGGCAGACAGACCA)mapstoCaMKIIβlinkerexon13.Variantsβ13,14b,16,17,18(44.8%),β13,14b,16,18(5.9%),β13,14b,17,18(4.0%),β13,14b,16,17,18-short-hub(3.7%)accountedforroughly58.4%ofmappedreadsfromtheCaMKIIβIlluminalibrary.Variantsinwhichexon14a(CAGCCAAGAGTTTAC)followedexon12,suchasβ14a,16,18(13.8%)andβ14a,16,17,18(9.1%),accountedforroughly30%ofmappedreads.Variantsinwhichexon14b(CCAAGAGTTTACTCA)followedexon12,suchasβ14a,16,17,18(7.1%),β14a,16,18(6.3%),andβ14a,17,18(1.2%)accountedforroughly14%ofmappedreads.Secondarypeakscorrespondingtoexons14aand14bareclearlyvisibleinthelast15bpoftheSangersequencingfragment.


https://doi.org/10.1101/721589

heterogeneity in human hippocampal camkii transcripts ... · 1 heterogeneity in human hippocampal...

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