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TranslationaltoxicologyandrescuestrategiesofthehERGchanneldysfunction:biochemicalandmolecularmechanisticaspectsKai-pingZHANG1,2,Bao-fengYANG1,2,Bao-xinLI1,2,*1DepartmentofPharmacology,HarbinMedicalUniversity,Harbin,China;2TheState-ProvinceKeyLaboratoriesofBiomedicine-PharmaceuticsofChina(KeyLaboratoryofCardiovascularResearch,MinistryofEducation),ChinaThehumanether-à-go-gorelatedgene(hERG)potassiumchannelisanobligatoryanti-targetfordrugdevelopmentonaccountofitsessentialroleincardiacrepolarizationanditscloseassociationwitharrhythmia.
DiversedrugshavebeenremovedfromthemarketowingtotheirinhibitoryactivityonthehERGchannelandtheircontributiontoacquiredlongQTsyndrome(LQTS).
Moreover,mutationsthatcausehERGchanneldysfunctionmayinducecongenitalLQTS.
Recently,anincreasingnumberofbiochemicalandmolecularmechanismsunderlyinghERG-associatedLQTShavebeenreported.
Infact,numerouspotentialbiochemicalandmolecularrescuestrategiesarehiddenwithinthebiogenesisandregulatingnetwork.
Sofar,rescuestrategiesofhERGchanneldysfunctionandLQTSmainlyincludeactivators,blockers,andmoleculesthatinterferewithspecificlinksandothermechanisms.
TheaimofthisreviewistodiscusstherescuestrategiesbasedonhERGchanneltoxicologyfromthebiochemicalandmolecularperspectives.
Keywords:potassiumchannel;humanether-à-go-gorelatedgene(hERG);longQTsyndrome(LQTS);activator;blocker;siRNA;biogenesis;proteintraffickingActaPharmacologicaSinica(2014)35:1473–1484;doi:10.
1038/aps.
2014.
101;publishedonline24Nov2014IntroductionThehumanether-à-go-gorelatedgene(hERG)potassiumchannel,alsoknownasKv11.
1,isencodedbytheKCNH2genelocatedonchromosome7q36.
1[1].
ThemainisoformofthehERGproteincontains1159aminoacidscomprising6transmembranesegments,aPer-Arnt-Sim(PAS)domainattheNterminusandacyclic-nucleotide-bindingdomain(cNBD)attheCterminus[1,2].
ThepotassiumcurrentconductedbythehERG(I[Kr])isessentialforrepolarizationofcardiomyo-cytesafteraheartbeat.
ReductionofI[Kr]canleadtolongQTsyndrome(LQTS),whichischaracterizedontheelectrocar-diogrambyprolongedQTintervalsandarrhythmia.
Infact,hERGplaysaroleinvariousphysiologicandpathologicprocessessuchascancer,digestive,secretoryandreproduc-tivesystems,signaling,diseasesofthenervoussystemaswellasdevelopment[3].
However,researchonhERGhasfocusedmainlyoncardiacrepolarizationandLQTS[3].
LQTSmayhaveseveremanifestationsincludingtorsadedepointes(TdP),syncope,andsuddencardiacdeath[4,5].
Type2LQTS(LQT2)refersspecificallytoasubclassofthediseasethatisduetogeneticmutationsinhERGthatunderlieapprox-imately45%ofallcongenitalcases[1,4].
ProlongationoftheQTintervalmayalsoresultfrommedicationsthatinhibithERGchannelsasasideeffectofthetherapeuticaction.
Therefore,theUnitedStatesFoodandDrugAdministration(FDA)hasdesignatedhERGasanobligatoryanti-targetindrugdevelop-ment.
ExistingdrugsmayalsobewithdrawnfromclinicalusebecauseoftheiradverseeffectsonthehERGchannel.
Well-knownexamplesincludeterfenadine,cisapride,andastem-izole.
Fordecades,themajorityofstudiesinhERGtoxicologyhavefocusedontheinhibitoryeffectsofvariouscompoundsonhERGchannelgating.
However,severalpuzzlesremainunsolvedwhenapplyingthechannelblockadetheory.
Forexample,arsenictrioxide,adrugusedtotreatacutepromyelo-cyticleukemia(APL),isfoundtocauseQTintervalprolonga-tionduringAPLtherapy[6].
ArsenictrioxideinhibitshERGchannelexpressionwithoutsignificantchannel-blockingactiv-ity[7].
Inaddition,wepreviouslydiscoveredthatceramidewasnotabletoalterhERGcurrentbyacuteapplication(superfu-sionfor25min)butrathercausedpronouncedhERGcurrentinhibitionafterprolongedincubationfor10h[8].
Anincreas-Review*Towhomcorrespondenceshouldbeaddressed.
E-maillibx64@hotmail.
comReceived2014-05-14Accepted2014-08-201474ingnumberofstudiesareshiftingtheirfocusfromchannelblockadetootheraspectsofchannelregulation.
Toxicologicalmechanismsareoftenfoundtofunctionwithinthebiochemi-calandmolecularaspectsofphysiologicalprocesses.
Indeed,recentyearshavewitnessedtheemergenceofadeeperunder-standingofthebiochemicalandmolecularfactorsunderlyinghERGregulationandthecorrespondingrescuestrategiesforhERGdysfunction.
hERGchannelbiogenesisisamultifacetedprocess.
ThehERGgene(KCNH2)istranscribedintomRNAinthenucleus.
ItisbelievedthatonlythemRNAthatissuccessfullypro-cessedthroughthenonsense-mediatedmRNAdecay(NMD)checkpointcanbetranslatedintheendoplasmicreticulum(ER)[9].
NascenthERGchannelsareformedintheERafterproperproteinfolding,subunitassembly,phosphorylation,andinitialglycosylation.
ThroughtheER-Golgiinterac-tioncomplex(ERGIC),partiallyglycosylatedhERGenterstheGolgiapparatusandundergoescompleteglycosylation.
Finally,thechannelissorted,carriedandinsertedintotheplasmamembrane[10].
Meanwhile,channelsalreadypresentontheplasmamembranearedynamicallyinternalizedandrecycled.
AlmosteveryaspectofhERGchannelbiogenesiscouldbepotentiallyaffectedbygeneticorpharmacologicalfactorsinamannerthatcontributestoLQTS.
Forexample,approximately30%ofheterozygousgenemutationsoftheKCNH2geneareprematurestopcodonsandsubjecttoNMD,whichleadstoa50%functionallossofthehERGchannel[1].
Pointmuta-tionsG601SandT473PresultinhERGtraffickingdeficiency,andT473PeveninducessevereTdPandcardiacarrest[11,12].
hERGproteinfoldingcanbedisturbedbydrugslikearsenictrioxide[7].
hERGmaturationandexportationfromtheERarearrestedbypentamidine,anantiprotozoaldrug[13].
More-over,degradationofthehERGproteincanbeincreasedbyceramide[14].
GiventhecloserelationshipbetweenhERGchanneldys-functionandLQTS,rescuestrategiesforhERGchanneldys-functionarebeingdeveloped.
Forexample,lowtemperature(27°C)iscommonlyappliedasarescuestrategybystabilizingtheconformationofthehERGchannel[15,16].
Inaddition,thehERGchannelblockerE-4031isabletoremovethetrafficking-deficientG601S-hERGchannelfromamicrotubule-dependentqualitycontrolcompartmentandincreaseexportfromtheER[17].
Meanwhile,thehERGchannelactivatorRPR260243canslowdownhERGchanneldeactivation,increaseI[Kr]andreversedofentilide-inducedactionpotentialduration(APD)andprolongation[18–20].
Rescuestrategiesarealsobeingdevel-opedfromthebiologicalregulatingfactorsofthehERGchan-nel.
OverexpressionofHsp90successfullyrescuesexpressionoftheA422T-hERGmutant[21].
ThisarticleprovidesanoverviewofknownmechanismsunderlyinghERGchannelregulation,withanemphasisonbiochemicalandmolecularcomponentsofhERGbiogenesisandpossiblerescuestrategiesforhERGdysfunction.
ChannelbiogenesisandregulatingnetworkThebiologicalregulationofhERGinthenucleusandERissummarizedinFigure1.
Figure1.
BiologicalregulationsofhERGinnucleusandatER.
TranscriptionofthehERGchannel(KCNH2gene)isundertheregulationoftranscriptionfactorSp1andtheNMDmechanism.
Hsc70andHsp70atERareresponsibleforearlyfoldingofhERGwhileHsp90inthecytoplasmisresponsibleforlatefolding.
Hop,DNAJA2,andFKBP38assisthERGreleasefromHsc70.
HopandFKBP38aidhERGtorecruitHsp90foritsmaturation.
Hsp40promotesdegradationofhERGintheproteasome.
1475TranscriptioninnucleusResearchonhERGchanneltranscriptionislimitedandcentersprimarilyontheNMDofprematuremRNA.
NMDpartici-patesmainlyingeneexpressionregulationandmRNAqualitycontrol.
Ithasbeenreportedthat30%ofKCNH2genemuta-tionsinduceNMD,whichisenoughtocausea50%reductionofhERGcurrenttherebydecreasingI[Kr][1].
Ithasalsobeenreportedthatanextensivelyexpressedtranscriptionalfactor,SP1,up-regulateshERGmRNA.
Furthermore,drugsmayalsorescuearsenictrioxide-inducedhERGdeficiencythroughup-regulationofSP1[22].
Thus,NMDandSP1mightbegoodentrypointsthroughwhichinterferenceofhERGdysfunctionmayoccur.
TranslationandtraffickingthroughERandGolgiapparatusTheinitialhERGproteinistranslatedintheER.
Theinitial,immatureproteinundergoesaseriesofcomplexmodificationsbeforebeingtransportedtothecellmembrane.
Generallyspeaking,nativehERGproteinisfoldedandassembledwiththeassistanceofchaperones[1,23].
Properlyfoldedandassem-bledhERGproteiniscoatedintheCOPIIvesicle,transportedforwardtointeractwithERGICandarrivesattheGolgiappa-ratuswhereitundergoesfull-glycosylation[1,11].
ChannelfoldingandassemblingregulationatERhERGchannelfoldingandassemblyareundertheregulationofacomplicatedcyclicchaperonesystem.
Amongthenumer-ouschaperones,Hsp/c70,Hsp90,Hop,FKBP38,Hsp40,DJA,14-3-3proteinandGM130havebeenbestcharacterized.
Heatshockprotein70kDa(Hsp70)anditscognate(Hsc70)participateinthefoldingofnativehERGwhile90kDaheatshockprotein(Hsp90)isinvolvedinthematurationoflate-phasehERG[23,24].
ItisbelievedthatHsp70andHsc70bindwithandregulatethehERGchannelinareciprocalway[25].
DNAJA2andHopassistinhERGreleasefromHsc70[26,27].
HopalsoaidsthehERGproteininrecruitingHsp90[27].
Hsp90effectivelyinhibitsmistakenly-foldedproteinaggregationandpromoteshERGmaturation[23].
FKBP38,a38-kDaFK506-bindingprotein,interactsbothwithHsc70andHsp90andmayalsobeaprerequisiteforhERGexportfromtheER[27].
Specifically,hERGdegradationisthoughttoberegulatedbyHsp40.
DJA1andDJA2,type-IofHsp40,candecreasetheinteractionbetweenhERGandHsc70thuspromotingearlydegradationofhERG[28].
AmorerecentstudydemonstratedthattheN-terminalCapofthehERGchannelisvitalforthestabilityofthePer-Arnt-Sim(PAS)domain,hERGassemblyandhERGtrafficking[29].
Importantly,drugscommonlyinterferewithchannelfold-ingandassembly,bothofwhicharealsotargetedforrescueofchanneldysfunction.
IthasbeenconfirmedthatarsenictrioxideinhibitsthehERGchannelbydisturbingitsinterac-tionswithHsp90andHsp70[7].
WhiletraffickingdeficiencyofA422T-hERGcanbecorrectedbyHsp90overexpression[21].
FKBP38hasalsobeenshowntopartiallyrestoretraffickingofF805C-hERG[25].
Therefore,channelfoldingandassemblyaremostlikelyadditionalpromisingentrypointsforinterferingwithhERGdysfunction.
ForwardtraffickingfromERtoGolgiProperlyfoldedandassembledhERGproteiniscoatedinCOPIIandtraffickedtotheGolgi.
ItisbelievedthatforwardtraffickingintheCOPIIvesicleisregulatedbyGTPaseSar1whilebackwardtraffickingintheCOPIvesicleisundertheregulationofGTPaseARF1[30].
Rab11BisanotherregulatingfactorthatinhibitsforwardtraffickingofthehERGchannel[30].
AttheGolgi,GM130isthecommonlyrecognizedchaperonethatregulateshERGchanneltrafficking.
GM130participatesinorientingthechanneltowardtheGolgibyinteractingwiththeCterminalofthehERGchannel.
However,GM130over-expressioncauseshERGcurrentreduction.
Furthermore,GM130mightactasacheckpointforhERGmaturation[31].
ItiswellknownthatmoststudiesonchanneltraffickingarefocusedontheER.
However,consideringtheimportanceoffullglycosylationofthehERGchannelattheGolgi,adeeperunderstandingofthecharacteristicsofthepatternofregula-tionattheGolgiisessential.
Moreover,hypoxia-induceddecreasedbiogenesisofthehERGchannelisreportedtoresultfromenhancedROSformationatthemitochondria[32].
TheseobservationssuggestthatthephysiologicalandpathologicalfactorstakingplaceintheGolgiandmitochondriaarepoten-tiallyofgreatclinicalsignificance.
QualitycontrolThetraffickingprocessisundertheregulationofastrict-monitoringmechanismcalledqualitycontrol(QC).
QCoccursmainlyintheER.
WhenthehERGproteinisunfoldedormisfolded,QCwillactivatetheunfoldedproteinresponse[UPR,alsocalledendoplasmicreticulumstressresponse(ERS)]topreventabnormalforwardtrafficking.
Specifically,chaperonesareoverexpressedtoassistproteinfolding[17].
Mistakenly-foldedhERGproteinisfatedtobemarkedwiththeC-terminaloftheHsp70-interactingprotein(CHIP)andubiquitin,anddegradedattheproteasomebytheendoplas-micreticulum-associateddegradation(ERAD)mechanism[1].
Forexample,G572R-hERGandE637K-hERGmutantsup-regulatetheactivatingtranscriptionfactor6(ATF6)-oneearlyresponderofUPR.
DownstreamtargetsofATF6,calnexinandcalreticulin,arefurtherincreasedtohelphERGchannelfold-ing[17].
QCisanessentialmechanismthatguaranteesthecorrec-tionandclearanceofabnormalchannels.
Understandingthismechanismwillhelptoclarifymorephysiologicalandpatho-logicalmechanismsandwillaidinthediscoveryofmoreres-cueapproachesforhERGdysfunction.
ThisissummarizedinFigure2.
EndocytosisandrecyclingoncellmembraneThehERGproteinthatsuccessfullyreachestheGolgiisthenfullyglycosylated.
Afterwards,thechannelissorted,sub-locatedandfinallytransportedtothecellmembrane.
There,thehERGchannelisdynamicallyinternalizedintothecyto-plasmandrecycledbacktothemembrane[1,23].
1476Itisknownthatmanytransmembraneproteinslackcyto-plasmicsequencesthatarerequiredforrecruitmentandinter-nalizationintoclathrin-coatedvesicles[33].
Caveolin(Cav),dynaminandArf6arethemostcommonlyreportedmembersofclathrin-independentproteinsthatregulateproteininternal-ization,includinghERGchannelinternalization.
Caveolin-3(cav3)andcaveolin-1(cav1)havebeenreportedtoco-localizewithhERGproteinonthecellmembraneandregulatechannelinternalization[14].
Inaddition,ithasbeenshownthatcaveolarendocytosis,involvingthecaveolin,isdynamin-dependent[33].
OnemostrecentstudydiscoveredthatthemembranalhERGproteinundergoesrapidinternalizationinanArf6-involvinganddynamin-independentmanner[34].
Thisprocessissumma-rizedinFigure3.
OnefateofinternalizedhERGproteinistobeubiquitinatedwiththeassistanceofubiquitinligaseandthendegraded.
Forexample,theWWdomainoftheubiquitinligaseNedd4-2caninteractwiththePYmotifofhERGchannel[35].
Co-localizationofCav3,Nedd4-2andhERGproteinaccelerateshERGubiquitinationanddegradationbycav3andNedd4-2,respectively[36,37].
AnotherfateofinternalizedhERGpro-teinistoberecycledbacktothemembrane.
Rab11isonememberthatassistsinthisrecyclingprocess[37].
However,ithasbeenreportedthatRab4overexpressionreduceshERGchannelexpressionbyreducingdegradationofNedd4-2[38].
Moreover,theserumandglucocorticoid-induciblekinase3(SGK3)andSGK1havebeenshowntonegativelyregulateNedd4-2-mediatedubiquitinationandpositivelyregulateRab11-mediatedrecycling[37].
Thus,themembranalhERGproteinappearstobeunderadynamicbalanceofdegradationandrecycling.
Notably,probucolhasbeenshowntospeeduphERGdeg-radationbyinfluencingcav1[39].
CeramideincreaseshERGdegradationbypromotingubiquitination[14].
Meanwhile,thetraffickingdeficiencyofthecysticfibrosistransmembraneconductanceregulator(F508-CFTR)iscorrectedbyknockingdownNedd4-2.
KnockdownofSGK1completelyblockedthisrescue[40].
Rab9isabletorescueprogesterone-inducedhERGchanneltraffickinginhibition[30].
Therefore,Rab9,SGK,andNedd4-2arepromisingcandidatesthatshouldbeexploitedforrescueofhERGchanneldysfunction.
OtherregulationmechanismsIthasbeenreportedthatprolongedAPDandinhibitedI[Ks]canbenormalizedbyanincreaseinI[Kr],supportingtheexistenceofcomplementaryinteractionsbetweenI[Ks]andI[Kr][41].
I[Kr]andI[Ks]arebothimportantcurrentsthatareresponsibleforcardiacrepolarization.
I[Kr]isconductedbyHERG(αsubunitofI[Kr])andKCNE2(βsubunitofI[Kr])whileI[Ks]isconductedbyKCNE1andKCNQ1[2].
IthasbeenshownthatthecurrentofKCNE2-HERGco-expressionbaresagreatersimilaritytoI[Kr]thansingleHERGexpressionandthatKCNE2isalsorelatedtoLQTS[42].
hERGcaninteractwithKCNQ1-αviatheirCOOHdomains[12]andtrafficking-sufficientKCNQ1canmodulatethehERGchannel[43].
Moreover,KCNE1isreportedtoco-immunoprecipitatewithhERG[44]andKCNE2mayreducehERGexpressionbyacceleratingdegradation[45].
Thus,inter-actionsamonghERG,KCNQ1andKCNEareessentialfortheirexpression[46].
Importantly,thepolymorphismsinthehERG/MiRP1K+-channelhasbeenreportedtopotentiallyFigure2.
QualitycontrolofhERGatER.
NormalhERGiscoatedinCOPII(regulatedbyARF-1)toforwardlytrafficktotheGolgiandinteractwithGM130.
AbnormalhERGiscoatedinCOPI(regulationbySar-1)andbackwardlytraffickedtotheER;accumulationofabnormalhERG(especiallyunfoldedhERG)willactivateUPR;ATF-6willshufflefromtheERtotheGolgi,ATF-6iscuttoformcleaved-ATF-6andtotravelintothenucleusandupregulatecalnexinandcalreticulintoassisthERGfolding;cATF-6can,however,promoteinteractionbetweenhERGandCHIP,thenhERGisrecognizedandmarkedwithubiquitintosignalitsdegradationintheproteasome.
1477increasesusceptibilitytoarrhythmia[47,48].
Clearly,futurestudiesareneededtoclarifytherelationshipsamonghERG,KCNQ1andKCNEproteinsandtheirintrinsicmodulatingmechanisms.
Ions,temperature,age,gender,andoxidativestressareotherfactorsthatregulatethehERGchannel.
Forexample,extracellularK+isaprerequisiteforhERGchannelfunctionandmembranestability[46].
LowextracellularK+willinducechannelmono-ubiquitination,anddegradationinthelyso-some[49].
Furthermore,internalizationanddegradationareacceleratedunderconditionsoflowextracellularK+[46,49].
Inaddition,itisbelievedthatintracellularK+helpstostabilizehERGconformationtherebyfacilitatingexportfromtheER[50].
hERGchannelbiogenesisrateandfunctionarebothdecreasedunderconditionsofanoxiaduetotheformationofROSinthemitochondria[51].
Notably,prolongedAPDofR190Q-KCNQ1canbenormal-izedbyhERGchannelinterventionandtrafficking-sufficientKCNQ1canalsorescuedecreasedG601S-hERG/F805C-hERGchannelexpression[41,43].
UnderstandingtheseregulatorymechanismsmayprovideusefulrescuestrategiesforhERGdysfunction.
hERGchannelandLQTSAtpresent,themostcommondiseaserelatedtohERGchanneldysfunctionisLQTS.
PatientswithaprolongedQTintervalaresusceptibletoarrhythmiaincludingtorsadedepointesandsuddencardiacdeath.
ThehERGabnormalityassociatedwithinductionofLQTSincludesacquiredLQTS(aLQTS)thatismostlycausedbydrugs(asshowninTable1)andcongenitalTable1.
MechanismsofhERGchannelinhibitionbyrepresentativedrugs.
MechanismtypesDrugnameDetailedmechanismsDirectblockDofetilide54HERGcurrent↓;blockatF656Propafenrone56HERGcurrent↓;structurewithnitrogen,hydroxy,rightangleTraffickingdefectAs2O37HERGcurrent↓;ICa2+-APD-QT↑;trafficking↓,Hsp90-herg↓Pentamidine13HERGcurrentandexpression↓;ERretentionDegradationCisapride58HERGcurrentandexpression↓;ubiquitination-degradationProbucol23HERGcurrentandexpression↓;Ikr-APD↑;cavintererenceMulti-pathwayFluoxetine62HERGcurrentandexpression↓;blockS6area;traficking↓Desipramine63HERGcurrentandexpression↓;block;trafficking↓;degrade↑Figure3.
EndocytosisandrecyclingofhERGoncellmembrane.
CaveolinandNedd4-2bothco-localizewithhERGonthecellmembrane.
InstabilityofcaveolinorNedd4-2-dependentubiquitinationincreasesendocytosisofhERG.
Caveolin-relatedendocytosisisArf6-dependentanddynamin-dependent.
Mono-ubiquitinizedandmulti-ubiquitinizedhERGisdegradedinthelysosomeandproteasome,respectively.
Rab11promotesrecylingofhERGbacktocellmembrane.
Rab4andSGK3bothdecreaseNedd4-2-dependentubiqtuinationofhERGandSGK3meanwhilepromotesRab11-dependentreclycingofhERG.
1478LQTS(cLQTS)thatiscausedbygenemutations(asshowninFigure4).
aLQTSofhERGchannelManyfactorsmayinduceaLQTSincludinghypokalemia,anoxiaanddiversedrugs[49,51,52].
Drug-inducedaLQTSisthemostcommontype.
DrugsliketerfenadinehavebeenremovedfromthemarketbecauseoftheadversecardiacsideeffectscausedbyhERGchannelinhibition.
Sofar,approxi-mately136drugs(www.
crediblemeds.
org;www.
QTdrugs.
org)havebeenreportedtoprolongtheQTinterval.
ItissurprisingthatsuchadiversecategoriesofdrugsarecapableofinhibitingthehERGchannelincludingantiarrhythmic,antipsychoticandantidepressantdrugs.
Forexample,berber-ineinhibitsthehERGchannelcausingAPDprolongation[53].
ArsenictrioxideprolongstheQTintervalbyinhibitinghERGchannelexpressionandincreasingthecalciumcurrent[7].
Theantidepressantdrug,desipramine,inducesQTprolongationthroughmultiplepathwaysthataffectthehERGchannel.
Therefore,thehERGchannelisthemostcommoncausefordrug-inducedaLQTS.
ThesedrugsimpactthehERGchannelmainlybydirectblockingandbyinterferingwithpathwaysinvolvedinbiogenesis.
hERGchannelblockinghERGchannelblockingisthenumberonereasonfordrug-inducedhERGchanneldysfunction.
ThehERGchannelcanbeblockedatdifferentsitesbydifferentdrugsmakingitimpor-tanttoanalyzetheblockingprocess.
Fromthepointofviewofdifferentbindingsites,Phe656andThr652arethetwocommonsitesfordrugbinding.
Forexample,dofetilidebindstoPhe656todecreasehERGcur-rent[54].
WhilemostdrugsblockthehERGchannelthroughPhe656andTyr652,othersitesarealsotargets.
TheblockingeffectofE4031anddofetilideonthehERGchannelincludesVal625,Ser624andThr623(nearthepore),Gly648andVal659(ontheS6domain),andS631A(betweendomain5and6)[54,55].
Whenconsideringdifferentblockingcompounds,com-poundswithnitrogen,hydroxyorarightanglecanmoreeas-ilyinhibitthehERGchannel[56].
Inaddition,anaromaticsidechainonTyr652andahydrophobicsidechainonPhe656areneededforcisapride-associatedhERGcurrentblocking[57].
Asdrugsprefertoformπ-πstackinginteractionswithTyr652andPhe656,Tyr652and/orPhe656mutationscouldreducedrug-associatedhERGinhibition[58].
ThehydrophobicityandaromaticcharacteratTyr623,Tyr652andPhe656arethoughttobethemainreasonsforblocking[59].
VanderWaals'forcepresentonthechannelsurfaceisalsonecessaryforblocking[59].
Inconclusion,Tyr652,Phe656,andTyr623arethemostessentialsitesforblocking.
Val625,Ser624,Ser631,Gly648,andVal659arealsocommonsitesforblocking.
TherearemanydifferentopportunitiesforblockingsuchasprotonizednitrogenforminghydrogenbondswiththeoxygenofcarbonylatTyr623,ahalfaromaticringinteractingwithπ–πstackingatFigure4.
SchematicsofLQT2mutations.
Eachgreenballrepresentsanaminoacid.
Eachpurple,orangeandbluepointrepresentsonemutation.
Inaddition,theorangepointsrepresentmutationsshowinghERGchanneltraffickingdeficiencyandthebluepointsrepresentmutationsbeingmonitoredbytheNMDmechanism.
1479Tyr652andthehydrophobicpartcanformingahydrophobicinteractionwiththebenzeneringatPhe656.
Newdrugsthatoff-targetthesebindingsitesmightbeonegoodsolution.
hERGchannelbiogenesisinterferenceBiogenesisdisturbance,especiallytraffickinginhibition,isanemergingreasonfordrug-inducedhERGdysfunction.
Arse-nictrioxidedisturbsthehERG-chaperoneinteractionsoastoimpedechanneltrafficking[7].
HigherlevelsofmiR-133andlowerlevelsofhERGexpressionarealsoassociatedwitharse-nictrioxide-inducedQTintervalprolongation[60].
PentamidinebindswiththehERGchanneltoarrestitsmaturationandinhibitsexportationfromtheER[13].
IthasbeenreportedthatpentamidinewithaphenylringorsubstituentsthatcanattachtothehERGchannelhasthelargestinhibitoryeffectonthehERGchannel[61].
DrugsalsoinfluencethehERGchannelbytheirimpactondegradation.
ProbucolspeedsuphERGchanneldegrada-tionthroughitseffectsonlow-densitylipoprotein(LDL)andcav1[23].
Ceramideincreaseschanneldegradationatthelyso-somebypromotingchannelubiquitination[14].
Infact,manydrugsinterferewiththehERGchannelthroughmultiplepathways.
FluoxetinebothblockshERGcurrentandreduceschanneltrafficking[62].
EvendesipramineinhibitsthehERGchannelbyblocking,traffickinginhibition,promotinginternalizationandacceleratingubiquitination-degradation[63].
AsthemajorityoftheprocessesinvolvedinbiogenesisofthehERGchannelareaffectedbydiversedrugs,rescuestrate-giesofhERGdysfunctionshouldbespecifictotheirtargets.
Forexample,drug-inducedtraffickingdefectscanbemini-mizedifcertainchemicalfeaturesareavoidedorremoved[61].
cLQTSofhERGchannelhERGchannelmutation-inducedcLQT2accountforapproxi-mately45%ofidentifiedLQTS[64].
Itwasreportedin2009that62%ofKCNH2mutationsweremissensemutations,24%wereframe-shiftmutations,and14%werenonsenseanddeletionmutations[65].
Upto90%ofmissensemutationsaffectchannelfunctionbytraffickinginhibition[66].
Mutationsarelocatedthroughouttheentirechannel:intracellulardomains(52%),trans-membranedomains(30%),poreareas(12%),andextra-cellularsegments(6%)[64].
CommonmutationcharacteristicsAsshowninFigure4,mutationsarelocatedthroughoutthehERGchannel.
Mutationsindifferentareashavediffer-entimpactsonthehERGchannel.
Intheporearea,G601SmanifestsasinhibitedtraffickingwhileY611Hmanifestsasincreasedubiquitinationanddegradation[11,67].
Thetrans-membranemutationT473PcausessevereTdPandcardiacarrest[12].
MutationsaroundT473PsuchasD456Y,F463L,N470D,andT474Iallmanifestastraffickinginhibitionwithdominantnegativeeffect[12].
Therefore,ithasbeeninferredthatmutationsbetweenS2andtheS2-S3linkerprobablyhaveseriousmanifestations.
MutationsatthePASdomaindonotfrequentlyinhibittrafficking.
IthasbeenshownthatK28E,F29L,G53R,C66G,L86RcausetraffickingdeficiencywhileN33T,R56Q,H70R,A78Rdonot[68].
N33TandR56QarelikelytodirectlyinfluencethemembranalhERGchannel,whileH70RandA78RprobablyindirectlyaffectthehERGchannelbyinteractingwithsurroundinghydrophobicareas[69].
TheredpointsinFigure4showthelocationsoftrafficking-deficientmutants.
Insummary,mutationsattheporeandtrans-membranedomainsprobablydisturbchanneltraffickingandhavesevereconsequences,whilemutationsatthePASdomainandtheCterminalarelikelytohavemilderconsequences.
RescueofthehERGchanneldysfunctionshouldconsiderthesetypesofcharacteristicsandavoidsuchmutations.
OthermutationcharacteristicsConsideringhERGchanneldysfunctionmechanism,somemutation-associatedhERGinhibitionsareregulatedbyQC.
Forexample,ATF-6,Grp78,Grp94,andcalreticulinareallactivatedtoassistinI593R-hERGfolding[68].
UPRisalsoacti-vatedbytheE637K-hERGandG572R-hERGmutations[17].
Inaddition,somemutationscanalterthekineticsofhERGchan-nel.
R744P-hERGcauseshERGchanneldysfunctionbyreduc-ingactivation[70].
T421M-hERGmanifestsasbothtraffickinginhibitionandchangesinkinetics[71].
Furthermore,mRNAisdecreasedviatheNMDmechanismforbothQ1070X-hERGandY652X-hERG[72,73].
Q725X-hERGandR1014X-hERGalsocauseaCterminalprematuretruncation[74].
Recently,astudydefinedtherequiredsegmentsforsusceptibilitytoNMD-associatedLQTS[75].
Regardingthetypeofmutation,thosethatexcludenon-sensemutationsalsoplayacertainroleincLQTS.
Frame-shiftmutationssuchasP1086fs+32X[76],splicingmutationssuchas2592+1G>A[77],andgenepolymorphismssuchasK897T,R1047LandQ1068RareallreportedtoinducecLQTS[76–78].
Specifically,patientscarryingtheA1116V-hERGmutationwithoutK897TgenepolymorphismmanifestnormalhERGfunction,whilepatientscarryingA1116V-hERGmutationandK897TgenepolymorphismmanifesthERGcurrentinhibi-tion[79].
Therefore,mechanismsthatexcludetraffickinginhibitionandmutationsthatexcludenonsensemutationscannotbeneglected.
Genescanningshouldbebroadenoughtoscreenoutdangerousfactors,especiallythosementionedabove.
CorrelationbetweenaLQTSandcLQTSResearchinvestigatingthecorrelationsbetweenaLQTSandcLQTSislimited.
OnestudydiscoveredDNAvariantsinapatientwhoexperienceddrug-inducedTdP[80].
ProbucolisalsofoundtoaccelerateM124T-hERG-associatedcurrentinhi-bition[81].
Furthermore,hERGgenepolymorphismisalsocor-relatedwithdrug-inducedhERGchannelabnormality[39,82].
Aspatientswhocarryriskygenemutationsaremoresus-ceptibletoLQTSwhentheyaretreatedwithpotentiallydan-gerousdrugs,itisnecessarytofurtherclarifythecorrelationsbetweenaLQTSandcLQTS.
WhenbothaLQTSandcLQTS1480aretakenintoconsideration,cross-linkrescuestrategyseemstoholdgreatpromise.
ExistingandpotentialrescuestrategiesAtpresent,rescuestrategiesforhERGchanneldysfunctionmainlyincludechannelblockers,activators,biogenesisregu-latingfactorsandothermethods.
AllrescuestrategiesaresummarizedinTable2.
Analysisofthemeritsanddemeritsofthesestrategieswillhelptodevelopthemostappropriaterescuestrategiesthatcanbeclinicallyapplied.
hERGchannelactivatorsActivatorsseemtobethemostpromisingstrategyforres-cuehERGchanneldysfunctionaswellasLQTSinvivo.
Sofar,thereareapproximately13reportedhERGactivators:RPR260243,PD118057,PD307243,NS1643,NS3623,ICA-105574,KB130015,A-935142,mallotoxin,matrine,oxymatrine,carbachol,andN-[N-(N-acetyl-L-leucyl)-L-leucyl]-L-norleu-cine(ALLN)[18,83–87].
ActivatorsprefertoregulatethehERGchannelbyalter-ingchannelkinetics[18].
Activatorsaredividedintoseventypesbasedontheirmechanismofactivation:(1)RPR260243mainlyaffectsthehERGchannelbyalteringdeacti-vation[18,88].
(2)PD118057,PD307243,NS1643,NS3623,andICA-105574allinfluencethehERGchannelbyregulatinginactivation[18,19,84,88–90].
(3)KB130015regulatesthehERGchannelbyincreasingactivation[18].
(4)A-935142andmal-lotoxinbothregulatethehERGchannelbychangingmultiplekinetics[20,83].
(5)MatrineandoxymatrineinfluencethehERGchannelbyup-regulatingtranscriptioninsteadofchangingkinetics[22,85].
(6)CarbacholincreasesNedd4-2phosphoryla-tion,slowsdownchanneldegradationandthusincreaseshERGchannelexpression[86].
(7)ALLN,aknowncalpain1/2andproteasomeinhibitor,rescuestheLQTS2phenotypebyrestoringtrafficking[87].
Importantly,manyactivatorsarecapableofshorteningpro-longedAPDandtheQTinterval.
Forexample,RPR260243isabletoreversedofentilide-inducedAPDprolongationbyslowingdownhERGdeactivationtoincreaseI[kr][18–20].
Inaddi-tion,PD118057canreducehERGinactivation,shiftupinac-tivationpotentialandpreventdofetilide-causedQTintervalprolongation[18,84].
However,PD118057failstocorrectthedominant-negativeeffectoftheE637K-hERG[91].
ICA-105574canalsodisablehERGinactivationandshortentheQTintervalincardiomyocytes[18,84].
Moreover,matrineandoxymatrinecanalsorescuearsenictrioxide-inducedhERGdeficiencyandAPDprolongation[22].
ALLNsuccessfullyreducesspontaneousarrhythmogenicepisodesandmarkedlyreducesAPD70/90ofLQTS-specificcardiomyocytesderivedfromhuman-inducedpluripotentstemcells[87].
Inconclusion,thehERGchannelactivatorsarepromis-ingagentstouseintherescueofdrug-inducedLQTSinvivo.
ActivatorssuchasRPR260243,PD118075,ICA-105574,andALLNshowthemostpotential.
MatrineandoxymatrinearealsopromisingfortherescueofothertypesofhERGdysfunc-tionowingtotheirroleinincreasingtranscription.
hERGchannelblockershERGchannelblockersarewidelyusedtorescuehERGdysfunctioninvitro.
E-4031,astemizole,fexofenadineanddofetilidearemostoftenchosen.
Forexample,E-4031isabletoremovetrafficking-deficientG601S-hERGfromthemicrotubule-dependentQCcompartmentandincreaseERexportofthemutanthERGchannel[17].
Inaddition,astemizoleandfexofenadinearebothreportedtorescueexpressiondefi-ciencyofN470D-hERG[15,92].
Dofetilideisreportedtocorrectpentamidine-inducedhERGdefects[61].
Nevertheless,therearequiteafewbarriersduetoblock-ersthatlimittheirapplication.
First,asblockersbindtotheintra-cavityofthehERGchannel,therescuedchannelmightbenon-conducting[1].
Forexample,underconditionsoflowintracellularpotassium,theastemizole-rescuedchannelisTable2.
Representativerescuestrategies.
RescuestrategyNameMechanismActivatorsRPR260243Deactivation↓P118057,PD307243,NS1643,NS3623,ICA-105574Inactivation↓KB-130015Activation↑A-935142,mallotoxinMulti-kineticsinfluenceMatrine,oxymatrineTranscription↑CarbacholDegradation↓ALLNRe-trafficking↑BlockersFexofenadineCompetivebindingE-4031,astemizolePromisingstrategiesFKBP38,Hsp90,Rab9Trafficking↑orrecyclingAMO,siRNA,iPSCSpecific-targetinginterferenceKCNQ1MutualinteractionKM57,KM60GlycosylationmodificationOthermethodsLowtemperatureConformatinstabalizationGlycerol,DMSO,TMO1481non-conducting[50].
Second,rescueconcentrationsofblockersareusuallyhigherthantheirblockingconcentrations,whichrestrictstheirapplication[92].
However,inthecaseoffexof-enadine,therescueconcentrationis350timeslowerthanitsblockingconcentrationonN470D-hERG[92].
Therefore,drugsthatarecapableofrescuinghERGdysfunctionandshowalowaffinityforthehERGchannelarepromising.
Third,therescueeffectsofblockersareoftenrelatedtotheirbindingsiteswithinthehERGchannelandthus,manytypesofLQTSarenotsensitive.
Hypoxia-inducedhERGtraffickingdeficiencyorT473P-hERG-associatedsevereTdPcannotberescuedbyblockers[14].
FexofenadineisalsounabletorescueV822M-hERGfunction[92].
Asaresult,hERGchannelblockersmaynotbethemostpromisinginvivostrategy.
However,fexofenadine'slowerrescuingthanblockingconcentrationmakesitapotentialres-cueofhERGdysfunctionclinically.
PotentialrescuestrategiestargetinghERGchannelNotsurprisingly,thereisgreatpotentialtodeveloprescuestrategiesfromfactorsthatregulatethehERGchannelbiogen-esis.
RegulatingfactorssuchasFKBP38,Rab9/11B,Hsp90,SGK,Nedd4-2,andKCNQarethemostpromising.
FKBP38promoteshERGtraffickingandisabletorescuetraffick-ingdeficiencyofF805C-hERG[27].
Rab9isalsoabletorescueprogesterone-inducedhERGtraffickinginhibition[30].
Fur-thermore,expressionoftrafficking-deficientA422T-hERGcanberescuedbyoverexpressingHsp90[21].
Finally,trafficking-sufficientKCNQ1canrestoreG601S/F805C-hERGchannelexpression[41,43].
Therescuingeffectsofthepreviouslymentionedstrategieslackspecificity.
SiRNAinterferenceandtheAMOstrategy,whichtargetsspecificobjectives,aredesignedtorescuethehERGchanneldefects.
Forinstance,E637K-hERG-targetingsiRNAreducestheexpressionofthemutantchannelwithoutaffectingexpressionofthewildtypechannel[93].
Allele-specificRNAinterference,whichtargetsc.
G1681A-KCNH2mRNA,successfullynormalizeshERGcurrentandAPD[94].
Moreover,pluripotentstemcells(iPSCs)bringgreathopeforrescuingofhERGdysfunction[95].
Althoughmostofthesestudiesareinvitro,becauseoftheirspecificity,regulatingfactorsandgene-targetingstrategiesarequitepromisingforclinicalapplication.
OtherrescuingstrategiesLowtemperature,glycerol,DMSOandTMOarecommonlyusedtorescuehERGdysfunction[15,16,23,69].
Temperature27°Cisoftenchosenforlow-temperatureincubation[15,16].
hERGchannelmutationssuchasK28E,F29L,D456Y,N470D,G601S,F805C,andV822Mandhypoxia-inducedhERGtraf-fickinginhibitionallcanberescuedbylow-temperatureincu-bation[23,69].
Chemicalchaperonessuchasglycerol,DMSOandTMOarebelievedtostabilizehERGchannelconformation[23].
Interestingly,chemicalsthatcanrescueotherabnormalitiesinotherchannelsarecapableofrescuinghERGdysfunctionaswell.
KM57andKM60,chemicalsthatcanrescueexpres-siondeficiencyoftheCFTRchannel,arealsoabletorescuetheG601S-hERGchanneldeficiency.
Specifically,KM57andKM60mightplaytheirrescuingrolebymodifyingglycosyl-ationofthehERGchannel[96].
Thisisespeciallyusefulwhenmorethanonetypeofchannelmutationoccurs.
Inaddition,wepreviouslyfoundthatacidificationmarkedlypotentiateddofetilide-inducedblockadeofthehERGchannelwhileitweakenedquiniding/azimilide-inducedhERGinhibition[97].
ConsiderationofbloodpHshouldhelptoreducedrug-inducedhERGchanneldysfunction.
ConclusionandprospectInsummary,hERGchannelbiogenesisandtheregulatingnetworkarequitecomplex,especiallyinregardtofunc-tionsoftheERanddynamiccyclingonthecellmembrane.
TherearemanybiochemicalandmoleculartargetsthatmayinducechanneldisordersincludingLQTS.
Inaddition,LQTS-associatedmutationsspreadthroughoutthewholeareaofthehERGchannel,particularlyintheporeareaandtrans-mem-branesegments.
Traffickingismostoftenaffected.
Rescuestrategiesincludeactivators,blockers,biogenesisregulatingfactorsandothermethods.
Lookingtowardthefuture,comprehensiveconsiderationandindividualtreatmentofthehERGchanneldisorderandLQTSarethetrends.
Activatorsseemtobethemostpromis-ingrescuestrategyforhERGchanneldysfunctionandLQTS.
Importantly,rescuestrategieswithgreatpotentialawaitexploitationinthebiogenesisregulationnetwork,especiallythetraffickingprocess.
AcknowledgementsThisworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(No31173050and30973530)andtheKeyProgramoftheNationalNaturalScienceFoundationofChina(No81230081).
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