Pharmacology of Voltage-Gated K Channels

PHARMACOLOGYOFVOLTAGE-GATED K+ CHANNELS

SarahBowden,ShukYinYeungandBrian channelitsextraordinaryselectivityandhigh Robertson throughputofpotassium(Figure1).Theflowrateof SarahBowdenisatIonixPharmaceuticalsLtd., K+ throughthesechannelsisveryclosetodiffusion, Cambridge,involvedinresearchingmechanisms anddrivenbytheelectrochemicalgradientforK+ ofpain,forthediscoveryofnovelanalgesicdrugs. acrossthecellmembrane. ShukYinYeungandBrianRobertsonareinthe NeuronalExcitabilityGroup,Biophysics,Biological Potassiumchannelscomeinavarietyofmodels.The Sciences,ImperialCollegeLondon,investigating simplestarecomprisedoffoursubunits,eachwith thephysiologyandpharmacologyofpotassium onlytwotransmembranesegmentseach(e.g.the channels,inanefforttobetterunderstandtheir inwardrectifiers).Others,recentlydiscovered,have functionalrolesinthemammalianbrain,andhow twoporeloopsfromeach subunit(whichmayhave K+ channelactivitymaybemodulated. eitherfouroreighttransmembranesegments);these arethe‘2-pore’K+ channels,whichprobably assembleasdimers toformthefunctionalK+ channel (seee.g.Goldsteinetal (2001)8 forreview). Introduction ThelargestclassofK+ channelsubunitsinmammals + Voltage-gatedpotassium(K)channelsarecrucial isthesixtransmembrane,singleP-loopchannels, regulatorsofmembraneexcitability,notonlyinthe andthesemaybeligand-orvoltage-gated(or CNSandheart,butalsoinmanyothertissues,such occasionally,both).Wedealherewiththevoltage- assmoothmusclecellsandwhitebloodcells.The fertilesymbiosisofelectrophysiologyandmolecular biologyhasproducedawealthofinformationabout thesecrucialproteins.Potassiumchannelsarethe Figure1.Theproposedarchitectureofvoltage- bestcharacterised,andmostdiverse,ofallion gatedpotassiumchannels. channelproteins.Wehavemany(butnotyet A. showsasingleα subunit.Thereare6putative sufficient)drugsavailablefortheirstudy,andmostof transmembranesegmentsS1-S6.Thepore-formingP thekeyplayershavebeencloned.Combininga loop,containingtheK+ ionselectivitysignaturemotif numberoftechniqueswith,forexample,specific ‘T/SxxTxGYG’,liesbetweensegmentsS5andS6 antibodylabelling,meansthatwearenowidentifying whiletheS4segmentisthepredominantvoltage- themolecularcorrelatesofnativevoltage-gatedK+ sensingdomain.B. showsthegeneralassemblyof currentsinalargenumberofmammaliancells. K+ channels.Theheteromultimericcomplexis composedoffour,Ploop-containingα subunits Thisverybriefoverviewisanattempttooutlinesome arrangedinatetramericfashion. ofthepharmacologicalpropertiesofvoltage-gatedK+ channels;theareaisvastandspacelimited,sothisis EXTRACELLULAR necessarilyarestrictedaccount.Theinterested -T/SxxTxGYG- readerisreferredtomorecomprehensive,excellent reviews(e.g.Coetzeeetal(1999),1 Shieh etal (2000),2 Coghlanetal (2001)3).Inaddition,theweb S1 S2 S3 S5 S6 providesseveralvaluablesources(e.g. P http://www.neuro.wustl.edu/neuromuscular/mother/ch an.html).Forafulleraccountofpotassiumchannel

structureandfunction,arecentreviewmaybefound N C inChoe(2002).4 ThoseinterestedinK+ channelsin INTRACELLULAR thecardiovascularsystemmaybedirectedtoArcher A. Asingleα subunitshowingtheK+ ionselectivitysignature andRusch(2001).5 Twosuperbtextbooksdealing motif‘T/SxxTxGYG’withintheporeloop withK+ channels’(andmostotherionchannels)

physiology,pharmacology,biophysicsanddisease S2 S1 S1 S2 67 S4 S4 areHille(2001)andAshcroft(2000). S3 S6 S6 S3 S5 S5 ThearchetypalK+ channelconsistsoffourprotein X subunits,eachofwhichcontributesitsownP-loopto S5 S5 S6 S6 S3 makethecompletefunctionalpore.Theporeloopisa S3 S4 S4 S2 S2 shortsegmentbetweentwotransmembrane S1 S1 segments;itdivespartlyintothemembrane,butdoes notfullycrossit.ThiscontainstheK+ channel B.ThegeneralassemblyoftheK+ channel;composedoffour, ‘signaturesequence’(T/SxxTxGYG),whichallowthe Ploop-containingα subunitsarrangedinatetramericfashion

TocrisCooksonLtd.,UK TocrisCooksonInc.,USA Tel:+44(0)1179826551 TollFreeTel:(800)421-3701Tel:(636)207-7651 Fax:+44(0)1179826552 TollFreeFax:(800)483-1993Fax:(636)207-7683 e-mail:[email protected] e-mail:[email protected] e-mail:[email protected] gatedK+ channels,inwhichoneofthe Kv1subfamilychannels transmembranesegments(S4)sensesvoltageshifts inthemembrane,andopenstheionchannel.The Kv1channelsarethemostdiverseinmammals,with focusofthisshortpharmacologicalreviewisthe severalmembers,numberedKv1.1to1.7.Dueto voltage-gatedK+ channelsintheKvfamily,EAG spacelimitations,andthefactthatanumberof family,andKCNQfamily(seeFigure2).Wewilldeal comprehensivereviewsareavailable(e.g.Coetzee et 132 withseveralmembersofeachfamily,knownas al(1999),Coghlanetal(2001),Shiehetal (2000)), subfamilies,inturn.Eachsubfamily(e.g.Kv1)may wewillfocusonlyonthosethathavebeenwell containanumberofmembers.Eachoftheseisthe characterisedphysiologicallyandpharmacologically. productofadifferentgene(e.g.Kv1.1),andwhen Duetothelargenumberoftheseimportantand expressedartificially,orinnativecells,canco- ubiquitousproteins,thereisnowagreatdealof assemblewithmembersofthesamesubfamily(e.g. informationabouttheirlocationinmammaliancells Kv1.1,1.2…).However,membersfromdifferent fromantibodylabellingstudies,and insitu subfamiliescannotco-assembletoformfunctional hybridization,andextensivedataontheirbiophysical channels.Whatthismeansfunctionallyhascrucial properties inexpressionsystems.Significantly,there implicationsbothforphysiologyandpharmacologyof areanumberofveryusefulpharmacologicaltools thefinalK+ channelexpressedinthecellmembrane. availabletoinvestigators.Wewilltreateachmember Aheteromultimericchannel(comprisedsay,1.1,1.2, inturn. 1.1,1.2,togiveasimple,andphysiologicallyrelevant example)willhaveahybridpharmacology,between Kv1.1subunits: Whenexpressedashomomers, Kv1.1(whichissensitivetoTEA)andKv1.2(whichis Kv1.1channelsbehaveasdelayedrectifiers,with insensitive,seebelow).Therefore,onemustexercise slowinactivation,andahalfmaximalactivation considerablecautionwhenextrapolatingfromthe voltageof~-30mV.TheyhaveanIC50 forTEA pharmacologicalliteratureforasinglechannelsubunit rangingfrom0.3to0.5mM,dependingonthestudy 10 toarealchannelinareal cellmembrane.Naturehas (seee.g.Grissmeretal (1994))andareblockedby complicatedthingsfurther,asK+ channelscan 4-aminopyridine(4-AP),withIC50’srangingfrom0.16 associatewithalargenumberof‘accessorysubunits’, to1mM.Ourownlabobtainedafigureof~150µM; whichcanalterthepropertiesoftheresulting thisagentblocksinthecationicformintracellularly 11 oligomericchannelevenmore(Figure2).Finally, (e.g.Stephens(etal 1994)).Agreatmanyother whencomparingdata,itmustbeborneinmindthat compoundsalsoblockKv1.1,butthemostusefulfor differentexpressionsystemscangivedifferentvalues dissectingoutthecontributionofthischannelsubunit forvoltage-dependence,ordrugpotency,for isofferedbynature’sownbesttools(developed example.Somefurtherdiscussionofthepitfallsmay throughthousandsofyearsofevolution),thetoxins. befoundinRobertson(1997).9

Figure2.Diversityofvoltage-gatedpotassiumchannels Theschematicshowsthemajorvoltage-gatedpotassiumchannelsuperfamilies,withthechannelsincludedinthis reviewinbold.Eachsuperfamilycomprisessubfamilies,eachofwhichmaycontainseveralmembers.Channels mayalsoassociatewithawidevarietyof‘accessory’subunits,includingKvβ andKChAPs(intracellularly)and MiRPs(transmembrane)whichcanprofoundlyalterthephysiologyandpharmacologyoftheresultingpotassium channelcurrent.

-T/SxxTxGYG-

S1 S2 S3 S5 S6

MIRP N C

KChAP

Kvβ

EAGKCNQSKslo Kv

KCNQ1 KCNQ2KCNQ3KCNQ4KCNQ5

eagergelkKv1Kv2Kv3Kv4 Kv5Kv6Kv7Kv8Kv9

2 Dendrotoxins(DTXs),afamilyof~7kDahomologous However,itismoreeffectiveatblockingKv1.3 polypeptides(approximately60aminoacidslong) channels(seebelow,Grissmeretal (1994)10),and isolatedfromblackandgreenmambasnakes,are alsoinhibitscalcium-activatedK+ currents.KTXis selectiveandeffectiveblockersofKv1.1(andothers; isolatedfromthevenomofthescorpion Androctonus seebelow)subunits.BlockisinthelownM m.mauritanicius ,andsharessomesequenceidentity (sometimespM)range,andoccursthroughspecific tocharybdotoxin(CTX)andnoxiustoxin(NTX).BgKis residuesintheK+ channelporeregion(Hurst etal apeptidefromtheseaanemone Bunodosoma (1991)12,Tytgatetal(1995)13).Dendrotoxins,with granulifera,whichblocksKv1.1,Kv1.2,andKv1.3 theirstrongoverallpositivecharge,bindtokey potassiumchannels(Alessandri-Haberetal (1999)20). negativelychargedaminoacidsneartheion Thereisagrowingnumberoftoxinsbeingidentified conductionpathway,namely:Kv1.1: thatblockKv1.1subunits,butmanyofthese,unless ….AEEAESH…..(forKv1.2:…..ADERDSH…..;for proteinengineered,alsoblockothermembersofthe Kv1.6:…..ADDVDSL……seebelow)(seeHurstetal Kv1-subfamily,sowewillnotdwellonthese.The (1991)12;NB,theseareforrat,andspecies interestedreaderisalsoreferredtoarecentattempt differencesdoexist).Amorerecentstudyhas torationalisethenomenclatureofthewholeraftof elaboratedtheprecisebindingbetweenthistoxinand peptidesisolatedfromscorpionvenoms(Tytgat etal theionchannelinmoleculardetail(Imredyand (1999)21). MacKinnon(2000)14).EachKv1.1toxin-sensitive channelsubunitiscapableofbindingDTX,buthigh Kv1.2subunits: Thesearealso‘delayedrectifier’in affinitybindingrequiresallfourindividualsubunitsto type,andbearmanysimilaritiestoKv1.1subunits, interactsimultaneouslyforhighaffinitybinding(Tytgat withwhichtheyareoftenco-associatedin etal (1995)13).Bindingis‘off-centre’,andDTXdoes heteromultimericcomplexes(e.g.inthesynaptic notappeartocaptheK+ channelpore(Imredyand terminalsofGABAergicbasketcellsinthecerebellar MacKinnon(2000)14).ThereareanumberofDTX cortex;Rhodesetal (1996)22,Southanand homologuesavailable(αβγδ-,-,-,-dendrotoxin I Robertson(1998)23).However,duetothelackofthe anddendrotoxinK).δ-DTXandtoxinKareclosely specifcTEA-bindingtyrosineresidueattheendofthe related(55outof57identicalresidues)andareused P-region(valineinKv1.2,Kavanaughetal (1991))24 as‘selective’blockersforKv1.1subunits(Robertson thesesubunitsareeffectivelyresistanttohigh etal(1996)15,Hopkins(1998)16),sincemassively concentrationsofexternalTEA.4-APblocksinthe higherconcentrationsareneededtoseeblockofthe sameconcentrationrangeasKv1.1channels.These otherDTX-sensitivesubunitsKv1.2and1.6(see subunitsareblockedbyα-DTXandDTX-I(figures below).Blockofnativecurrentsinrealcellsbyd-DTX fromHopkins(1998)16 showDTX-Ibeing~7times ortoxinKisnowbeingusedasadiagnosticforthe morepotentonKv1.2).AlsousefulininhibitingKv1.2 presenceofKv1.1channelsubunits(e.g.Southan istityustoxin-Kα,fromtheBrazilianscorpion Tityus andRobertson(2000)17,Hattonetal (2001)18). serrulatus,whichblockswithanIC50 of105pMfor mouse,but550pMforhumanKv1.2(Hopkins Maurotoxin(MTX)isa34-aminoacidpolypeptide (1998)16)andcharybdotoxin,whichblocksinthe cross-linkedbyfourdisulphidebridgesthathasbeen range1.7-17nM(Grissmeretal(1994)10,Coetzee et isolatedfromthevenomoftheTunisianchactoid al (1999)1).Thelatterhowever,alsoblockscalcium- scorpionScorpiomauruspalmatus. ItblocksKv1.1 activated(BKandIK)channelsandKv1.3subunits. andKv1.3channelsexpressedinXenopus oocytes Veryrecently,Dudinaetal (2001)25 haveprepared withIC50’sof40and150nMrespectively,butismore andidentifiedaninterestingnewselectiveinhibitorof effectiveagainstKv1.2subunits(IC50 =0.8nM) Rochatetal.(1999)19 AnotherusefultoolforKv1.1 Kv1.2channels.OsK2,a28aminoacidpeptidefrom theCentralAsianscorpion,Orthochirusscrobiculosus channelsubunitsiskaliotoxin(KTX,IC50 ~40nM). blocksKv1.2inoocyteswithanIC50 of97nM,and + hasnoeffectonKv1.1or1.3channelsat250nM. Figure3.ChemicalstructuresofsomeKchannel Thiswillbeanincrediblyusefultool,complementing blockers theKv1.1-selectiveDTX’sinelucidatingthefunctional rolesofthisimportantchannelsubtypeintheCNS. Indeed,Kv1.2isprobablythemostabundantKv1 subunitinthemammalianCNS.

Kv1.3subunits: Therehasbeenaconsiderable 4-AP(Cat.No.0940) interestinthephysiologyandpharmacologyofthis protein,sinceitplaysacrucialroleincellular proliferationinhumanTlymphocytes(e.g.Cahalan andChandy(1997)26).Inexpressionsystems,Kv1.3 CP339818hydrochloride(Cat.No.1399) channelshaveahalf-activationvoltageof~-30mV andshowmoderateinactivation(Coetzee etal (1999)1).TEAsensitivityis10-50mM,and4-AP blocksinthesamerangeasKv1.1and1.2.These channelsareresistanttothedendrotoxins,butare blockedbymargatoxinintherange1-10nM,andthis toxinhasprovedausefultoolinwide-rangingways, forthemappingofKv1.3residues,andin immunosuppressantstudiesinmammals. Other Charybdotoxin(Cat.No.1087) scorpiontoxinsusefulininhibitingKv1.3subunits includethosefromPandinusimperator venom(Pi1,2 and3,Peteretal (2000)27,(2001)28),and Centruroideslimbatus venom(Hongotoxin-1, Koschaketal (1998)29).Manymorearelikelytobe discovered.

ForblockersofKv1.3tobeclinicallyeffective,small (-)-[3RS,4]-Chromanol293B(Cat.No.1475) molecules,selectiveforthesechannels,havetobe designedordiscovered.Correolide,isatriterpine

3 naturalproductfromthetreeSpacheacorrea(Felix et Kv1.5channels,incommonwithmanyothers,are al (1999)30),whichisahighlypotentblockerofKv1.3 blockedbyinternalTEA(Fedidaetal (1996)44)acting + channelsexpressedinCHOcells(IC50 ~80nM) atasiteontheinnervestibuleoftheporeoftheK althoughatµMconcentrationsitblocksother channel.Verapamil,acalciumchannelblocker,also membersoftheKv1subfamily(Vianna-Jorge etal inhibitsKv1.5currents(Rampeetal (1993a)45), (2000)31,Cheongetal (2001)32).Otheragents,such probablybyanopenchannelblockingmechanismat asUK-78,282blockKv1.3withanIC50 of~200nM theinnerpore.Nifedipine,anothercalciumchannel byinteractingwithC-typeinactivation(Hanson etal (L-type)antagonistblocksKv1.5(Zhang etal (1999)33;notehowever,thattheseauthorsfoundthat (1997)46).Thresholdeffectsontheclonedchannel thiscompoundalsoblockedKv1.4channelswith wereseenat500nM,wellwithintheclinicalrange similarpotency).OtherblockersincludeWIN-17317-3 (ZhangandFedida(1998)47).Nifedipinecausesa andCP339818.However,theformerinhibitssodium voltage-andtime-dependentblock,and‘crossover’of currentsinCHOcellsstablytransfectedwiththerat thedeactivatingtailcurrents,buthaslittleeffecton brainIIAsodiumchannelwithhighaffinity(K=9i nM; gatingcurrents,andresultsfromdifferentrecording Wanneretal (1999)34)andalsoblockssomecalcium- configurationssuggestapreferentialblockatthe activatedpotassiumchannels.CP339818alsoblocks extracellularsideofthechannel(orabindingsitethat C-typeinactivatedchannelswithanIC50 valuearound ismoreaccessiblefromtheextracellularside). 200nM,butagain,blocksKv1.4channels(Nguyen et al (1996)35). Quinidineisanantiarrhythmicagentwhichblocksa varietyofK+ currents.ItrapidlyblocksKv1.5 Kv1.4subunits: Inhomomericassemblies,these channels,bindinginternallytoasitethatisexposed formarapidlyinactivating‘A-type’current.Their onlywhenthechannelopens.Oncequinidineis thresholdisaround-50mV,withhalfactivationat boundhere,itimmobilizesgatingcharge,whichonly ~-22mV.Theyhaveamarkeddependenceon returnsoncethedrugdissociatesfromitsbindingsite. external[K+],ascurrentscarriedbyKv1.4disappear Fedida(1997)48 suggeststhatincontrasttomany whenexternalpotassiumisabsent(Pardo etal otherblockingdrugs(e.g.4-AP),quinidineisapure (1992)36).Anotherusefuldiagnosticisthefactthat openchannelblockingagent,devoidofactionson Kv1.4subunitsareinhibitedbyfairlymildacidosis(pH closedchannels(ChenandFedida(1998)49). 6.5),duetoaslowingofrecoveryfromN-type inactivation(Claydonetal (2000)37).Kv1.4 Kv1.5channelsareblockedbybothflecainideand homomericchannelsareresistanttoexternalTEA, clofilium,butotherchannelsareprobablytheprimary butblockedby4-APintheconcentrationrange0.7to pointofclinicalactionofthesecompounds. 1 13mM(IC50’s,seeCoetzeeetal (1999)).4-AP Loratadineisanon-sedatingantihistamine,whose bindingrequireschannelactivationandhasmarked usemay,insomecases,causecardiacarrhythmia. usedependence(YaoandTseng(1994)38,Tseng Delponetal(1997)50andLacerdaetal (1997)51 have (1999)39).RemovingasubstantialpartoftheN- shownthatloratadineblocksKv1.5atconcentrations terminal(aminoacids3-25),whichremovesN-type inthehighnM-µMrange,reducingtheprobabilityof inactivationfromKv1.4,permits4-APtoblockmore openingofsingleKv1.5channels,andaccelerating 39 potently(IC50 =0.16mM,Tseng(1999)).Asimilar thedecayofmacroscopiccurrents.However,these interactionbetweenN-terminalinactivationand4-AP concentrationsareconsiderablyabovethoseseen blockwasalsoobservedforKv1.1channels therapeutically(Delponetal (1997)50).Twoothernon- (Stephensetal (1994)11).Thisagainunderscoresthe sedatingantihistamines,terfenadineandebastine, pointthatinheteromultimericassembliesofchannel havebeenexaminedonKv1.5(Rampe etal subunits,pharmacologicalbehaviourcanbemodified (1993b)52,Crumbetal(1995)53,Valenzuela etal notonlybydifferentdrugbindingsitesbeing (1997)54).TerfenadineblocksKv1.5inavoltage-and incorporatedinthefinalchannel,butalsodifferent time-dependentmanner;againhowever, kineticbehaviouroftheheteromultimericchannel. concentrationsrequiredarehigh(µM)comparedto ThereisnoblockbyDTX,oranyothertoxinsyet plasmalevels(lownM),andebastineshowedalmost tested.Xuetal (2001)40 haveshownthatthe noblockatallat3µM.RampeandMurawsky neuroprotectiveagentriluzoleinhibitsKv1.4channels (1997)55 havesuggestedthattheantibiotic withanIC50 of70µM,possiblythroughanoxidative erythromycin,atconcentrationsachievedfollowing mechanismontheN-typeinactivationdomainofthis intravenousinjection,blocksopenKv1.5channels channel. fromanintracellularsite,again,perhapscontributing toprolongationofcardiacrepolarizationseeninthe Overall,thereisashortageofselective,high-affinity clinicwiththisdrug. pharmacologicaltoolsavailableforKv1.4channels. Thisisapity,sincetheyarecriticallyimportantin Propafenone,anditsmajormetabolite5-OH- certainregionsoftheCNS,andarecentstudyshows propafenone,blockKv1.5,withIC50’sof~4and9Mµ thattheyaretheonlyKv1subfamilysubunit respectivelyat+60mV.Propafenoneblocksavariety expressedinsmallerdiameter‘pain’neurones ofcardiacvoltage-gatedchannels,includingNa+ and (Rasbandetal (2001)41). Ca2+ types,andisusedasanantiarrhythmic.Blockof Kv1.5istime-andvoltage-dependent,andresultsare Kv1.5subunits: Kv1.5isunquestionablythemost consistentwithopenchannelblock(Franqueza etal importantKv1-subfamilymemberinthehumanheart. (1998)56). TherapidgatingofKv1.5,andotherproperties(see Fedidaetal (1993)42,(1998)43 foracomprehensive Kv1.6subunits: Thesesubunitsalsobehaveas review)stronglysuggestthatthischannelsubunit ‘delayedrectifiers’inhomomericassemblies, makestheultra-rapidlyactivatingdelayedrectifierIkur activating~-50mV,withhalfactivation~-20mV. inthemammalianatrium.Ikur andKv1.5aresensitive TheyaresensitivetoexternalTEAinthelowmM torelativelylowconcentrationsof4-AP(Fedida etal rangeand4-APwithIC50’sbetween0.3and1.5mM 42 1 (1993),IC50 ~50µM).4-APcanblockopenand (seeCoetzeeetal (1999)).Kv1.6subunitsalsohave closedchannels,reducethegatingcharge(the the‘DTX’acceptorsite(seeaboveforsequence), conformationalchangesprecedingfinalchannel beingblockedinthelownMrange.Charybdotoxin, opening,Fedidaetal (1996)44,andslowC-type margatoxinandhongotoxinalsoinhibitKv1.6inthe inactivationinKv1.5(Fedidaetal (1996)44).Kv1.5 lownMrange(seeCoetzeeetal (1999)1).Another channels,likeIkur,areinsensitivetoexternalTEA scorpiontoxin,tamulustoxin(fromtheIndianred (onlytinyblockattensofmM).

4 scorpion,Methobuthustamulus)hasbeenrecently timesmore4-APtoblockthanKv3.1.Kirsch etal identifiedasablockerofKv1.6channelsexpressedin (1993)69 madechimericchannelconstructsinwhich CHOcells(Strongetal (2001)57). segmentsofKv2.1andKv3.1wereswapped;briefly, muchofthe4-APsensitivityresidesinthelatter, Kv1.7subunits: Kalmanetal (1998)58 have intracellularendofS6segment.Akeyareaofinterest expressedKv1.7channels,whichformafairlyrapidly isintheproposedoxygensensitivityofKv2channels, inactivatingcurrent,andidentifiedthisintheheartby asKv2.1isinhibitedbyhypoxia(PO2 =30mmHg), Northernblotting.Itisblockedby4-AP(IC50 ~250 andthischannelalphasubunitisfoundinsmooth µµM),tedisamil(IC50~18M)andnifedipine(IC~50 musclecellsofthecirculatorysystem(e.g.Hulme et 13µM).ItisinsensitivetoexternalTEA(ithasa al (1999)70).However,othersubunitsfromother hydrophobicalanineatthe‘TEAsite’),butispotently subfamiliesarealsoinhibitedbyoxygenshortage. blockedbytheStichodactyla seaanemonetoxinand noxiustoxin. Kv2channelsprovideanextralevelofinterestfor biophysicistsandphysiologistsinthattheymayhave TheKv1α-subunitscanhavetheirphysiological theirbehaviourmodifiedbyaccessoryα (sometimes propertiesalteredthroughassociationwith calledγ)subunits,which,whenexpressed intracellular,accessoryKvβ subunits(Castellino etal themselves,areelectrophysiologically‘silent’.Several (1995)59,Moralesetal(1995)60,Englandetal (1995), differentsubunitsareknowninthisclass:Kv5.1, Majumderetal (1995)62).Thenumberofβ-subunits Kv6.1,Kv8.1andKv9.1-9.3.AssociationofKv6.1with identifiedgrowsquicklyandthusfar,theyhavebeen Kv2.1slowstheclosurerateofKv2.1severalfold showntomodulateα-subunitsinanumberofways. (Postetal (1996)71),andshiftsactivation~-34mV Forexample,byincreasinginactivationrate,through leftwards.Furthermore,sinceKv6.1hasa contributinganN-terminal‘inactivationball’oftheir hydrophobicvalineresidue(insteadofthetyrosineof owntotheoligomericcomplex(Rettigetal (1994),63 Kv2.1)intheexternalTEAbindingsite,TEAblocking Englandetal (1995)61).Theymayalsoshiftthe potencyisabout10-foldlowerintheresulting activationcurveandotherelementsofchannelgating heteromultimericchannel.Kv6.1subunitsarequite (seee.g.Uebeleetal(1996)64,Heinemann etal commoninSAnodalcellsintheheart(Brahmajothi et (1996)65),andby‘chaperoning’channelstothe al (1997)72),suggestingthatthissubunitmightbe surfacemembrane(Shietal (1996))66 .Whilethereis physiologicallyrelevant.AnotherKv2-modifying notspaceheretofullydealwiththefurtherlevelsof subunitisKv8.1,whichcanslowactivationand complexityandsophisticationbestowedonKv1-α inactivationofKv2channelsquitedramatically subunitsbyβ-subunits,somekeypointsmaybe (Salinasetal(1997)73,Castellanoetal (1997)74). given.Associationwithβ-subunitscandramatically Kv9.1subunits,alsosilent,suppressexpressionof alterthephysiologicalprofileofKv1channels,making Kv2.1subunitsby~70%,andshifttheactivation otherwisenon-inactivatingchannelsrapidlyinactivate, curvetotheleft(RichardsonandKaczmarek andshiftgating.Thiswillmakeeasyextrapolationof (2000)75). behaviourandpharmacologyfromclonedchannelsin expressionsystemstonativecurrentsdifficult. TheKchAPsubunitsalsointeractwithKv2proteins, Additionally,changesingating,especially enhancingchannelexpressionbyactingasa inactivation,canaltertheresponsivenessofacurrent ‘chaperone’(e.g.Kuryshevetal (2001)76),butthereis toadrug.Ifthedrugshowsusedependence,thenN- littledetailyetonwhetherthesesubunitsalter typeinactivationmaychangethe‘access’toitssiteof pharmacologicalproperties. action.Also,cellsmayequallywellchangethe amountofβα-subunitsassociatingwiththeir Kv3subfamilychannels partnersunderdifferentphysiologicalor Kv3channelshaveanumberofinteresting pathophysiologicalstates. physiologicalandpharmacologicalproperties,and theirkeyrolesinmammalianCNSneuroneshave Kv2subfamilychannels precipitatedagooddealofinterestinthem.Thiswill Kv2(mainlytwoclassesKv2.1andKv2.2channels) onlybeabriefoverview,sinceanumberoffine, areactivatedatlowvoltagethresholdsandshowlittle comprehensivereviewsareavailable(e.g.Rudy etal inactivationduringvoltagepulseslessthan1second. (1999)77,RudyandMcBain(2001)78).Briefly,Kv3 Kv2.1expressioniswidespreadthroughouttheCNS, channelshaveunusualelectrophysiological andinhippocampus,islocalisedonsomataand propertieswhichallowthemtoplayspecialrolesin dendritesofpyramidalcellsandinterneurones, highfrequency,repetitivefiring.Theyareoftenfound formingamajorcomponentofthe‘delayedrectifier’ infastspikinginterneuronesinthecortexand potassiumcurrent.Kv2subfamilychannelsarealso hippocampus,aswellasintheauditorysystemand foundintheheart,wheretheymayalsobe thalamus. associatedwithaccessoryproteins(seebelow). Kv3channelsactivateatverydepolarisedpotentials, ThesedelayedrectifierchannelsaresensitivetoTEA morepositivethan-20mV,whichquitesetsthem and4-APblock,intherange4-10mMfortheformer apartfromallotherKvchannels.Theyactivate and0.5-5mMforthelatter(e.g.Coetzeeetal (1999)1 relativelyquickly,butdeactivateextremelyrapidly forreview).Theyarealsoinhibitedbythetoxins (lessthan1msatrestingpotentialatroom knownashanatoxins,isolatedfromvenomofChilean temperature).Kv3.4showsrapidN-type(conferredby tarantulas.InanelegantstudySwartzandMacKinnon a‘ball’initsN-terminus)inactivation,whilstKv3.2and (1995)67,(1997)68)showedthattheactivationcurve Kv3.1showlittleinactivation.Reportsshowvariable forKv2.1wasshiftedinapositivedirectionby kineticsforinactivationofKv3.3channels,which hanatoxin,andthatblockandshiftwerebest showtransientcurrentsinoocytes,butshowmuch explainedbyamechanismwherebyhanatoxinbinds lessinactivationwhenexpressedinmammaliancell totheS3/S4linkerregion,inhibitingchannelopening lines(seeRudyetal (1999)77 andcompareRudyand untilsubstantiallydepolarisedpotentialsarereached. McBain(2001)78).Onceagain,thisdemonstratesthat Overall,however,thepharmacologyofKv2channels weshouldexercisecautionwhenextrapolatingthe issomewhatsparse,butsignificantdetailisavailable kineticsofahomomericchannelincertainexpression forthesite(s)ofblockofTEA(internalandexternal) systemstothebehaviourofaK+ currentin‘native’ and4-APfromtheelegantworkofKirschand cells.MammalshavefourKv3genes,whichundergo colleagues.Forinstance,Kv2.1requiresabout100 extensivesplicing,yieldingsomeinteresting

5 differencesintheproperties(regulationbysecond wasnotuntil1998thatDiochotetal reporteda messengersystems,andcellularlocation,butnotin selectivepeptidictoxinblockerfortheKv3sub-family. biophysicalproperties)ofthechannelα-subunit Thistoxin,originallyknownasblooddepressing proteins(Rudyetal (1999)77). substance(BDS),wasisolatedfromthesea anemone,Anemoniasulcata andexistsintwo AswithmanyotherKvchannels,thereareveryfew isoforms,eachbeing47aminoacidsinlength,but selectivepharmacologicaltoolsthatcanbeusedto differingattworesidues.Theseauthorsshowedthat unambiguouslyidentifyKv3channels.However,they bothisoformseffectivelyandselectivelyblockedthe dohaveanextraordinarilyhighsensitivitytothe rapidlyinactivatingKv3.4channelwhenexpressedin ‘classical’blockersTEAand4-AP,andthese Xenopus oocytesandamammaliancellline(COS 79 compoundsareoftenusedtoprovidecompelling cells),withanIC50 of~50nM(Diochotetal (1998)). evidencethatnativecurrentshaveaKv3component. ThetoxinssharenosequencesimilaritiestootherK+ Kv3channelsareblockedbyexternalTEAwithan channeltoxins,anddonot blockotherKvchannels. IC50 of~200µM.1mMTEA,whichblocksKv3 BDSIandBDSIIarealreadybeingemployedby channelsalmostcompletely,onlyreallyhasdramatic investigatorsasselectivetoolsforKv3.4channel effectsonKv1.1homomericchannels,BKchannels, subunits,albeitatmuchhigherconcentrations(e.g. andKCNQ2channelsubunits,whichareeasily Riazanskietal (2001)80;2.5µMinbrainslices). distinguishedfromKv3channelsbytheirgatingand However,datafromourownlabsuggeststhatsome singlechannelconductance.Thishighsensitivityto circumspectionmightbeneeded.BDSIandBDSII TEAisduetothepresenceofatyrosineresidueat causeasubstantialblockofKv3.1andKv3.2currents theC-terminalendoftheP-region(presentinall at500nMinmammaliancelllines(seeFigure3); Kv3’s,aswellasKv1.1,1.6,2’s).4-APalsoblocks preliminarydata(Yeungetal,unpublished)suggests Kv3’s,withIC50’srangingfromthephenomenallylow thatthesetoxinsmightbeinhibitingKv3channelsby (20µM),upto0.6mM.Althoughthehighpotencyof shiftingtheactivationcurvetotheright,inasimilar 4-APblockisuseful(combinedwithotherevidence) mannertohanatoxin (seeabove).Furthermore,in tosuggestinvolvementofKv3channels,itis oneCNSslicepreparationthathasagooddealof importanttonotethatthisisa‘messy’compound, Kv3.4proteinasdeterminedbyantibodylabelling, withcomplexstateanduse-dependence.Valuesfor thereislittlesignoffunctionaleffectsofBDSIIat1 blockvarydependingalsoonexpressionsystem µM(Morrisetal,unpublished). usedandtimeofexposure. Kv3.1bchannelsubunitsareinhibitedbyhypoxia,and Untilfairlyrecently,therewerefewotheruseful, therebymaycontributetoO2 sensinginpulmonary selective,pharmacologicaltoolsforKv3channels.It vasculature(Osipenkoetal (2000)81).Thereisa growingliteratureonthemodificationofthegatingof Kv3channelsbyavarietyofsecondmessenger Figure4.WholecellclamprecordingsfromKv3 systems,includingPKA,PKCandPKG,whichis channelsexpressedinmammaliancelllines beyondthescopeofthepresentreview. Cellswereheldat-80mV;voltagestepsto+40mV Nevertheless,itisclearthatKv3channelsmaybe for100ms.Externalrecordingsolutionscontained35 + dramaticallymodulatedbyphosphorylation,which mMK.A. Kv3.2channelstransientlyexpressedin canhavemarkedeffectsonthephysiologyandgating modifiedHEK293cells.15minutesexposureto ofthecurrent(e.g.MacicaandKaczmarek(2001),82 500nMBDS-IIresultedin~50%reductionofthe Morenoetal (2001)83).Thistooshouldbebornein current.B. Kv3.1achannelspermanentlyexpressed mindwhenusingpharmacologicalandphysiological inamousefibroblastcellline(B82).Blockof~40% criteriaderivedfromexpressionsystems. uponexposureto500nMBDS-IIfor6minutes. 84 Recently,Abbottetal (2001)reportedanIC50 for BDS-IIof~250nMforKv3.4channelsexpressedin A CHOcells.Thischangeddramaticallyto~7µMwhen Kv3.4co-associatedwiththeaccessorysubunit MiRP2(Min-K-relatedpeptide2).Therearefew studies(sofar)oftheassociationofKv3channels withaccessory subunits,butthefascinatingstudyof Abbottetal showshowdramaticallyphysiologyand +BDS-II pharmacologyoftheα-subunitmaybechangedby suchco-association.MiRP2isonememberofthe KCNEgenefamily,whichmakeasmall,single transmembranepasspeptidethatassociatewithKv α-subunits(thebestknownexamplebeingMinK whichcombineswithKCNQ1channels,seebelow). MiRP2-Kv3.4channelshavedramaticallydifferent 2nA conductance,andvoltagegatingfromthe‘parent’ subunitalone.Indeed,combiningwithMiRP2shifts 20ms theactivationcurvealmost50mVtotheleft,and Abbottetal convincingly suggestthatthischannel assemblymightmakeacontributiontotheresting B membranepotentialinskeletalmuscle,asmutations onMiRP2leadtoperiodicparalysisinman.Stocker etal (1999)85 havealsoreportedthatKv9subunits– otherwiseelectricallysilent–candecrease expressionofKv3subfamilychannels.Itwouldnotbe +BDS-II surprisingifthereweremanymore‘accessory’ proteinsforKv3subunitswaitingtobediscovered.

Kv4subfamilychannels Thisimportantclassofvoltage-gatedchannel subunitsformsfast,transient(A-type)currentswhich

6 activateatmembranepotentialsbelowthresholdfor DataareavailableontheblockofKv4channelsby actionpotentialgeneration.Agreatmany moreconventional‘cardiac’blockers.Yeolaand sophisticatedexperimentsinvolvingantisenseand Snyders(1997)93 comparedthesensitivitiesofKv1.4 dominantnegativeconstructs,orothergeneproduct andKv4.2totwodrugs(quinidineandflecainide), eliminationmechanismshaveshownthatKv4 whichareeffectiveblockersofIto.Flecainideblocked subfamilychannelproteinsunderlieIto inmany Kv4.2(10µMblockedby~50%),buthadminimal cardiacmusclecells,aswellassomato-dendriticA effectsonKv1.4.Quinidinewasaboutequipotent currentsincentralneurones.Inneurones,these againsteachclone(10µMblockedby~50%). channelscanexertaprofoundinfluenceoninterspike interval,therebyregulatingthefrequencyofrepetitive Wangetal (1997)94 madethesurprisingfindingthat firing.Theyareespeciallyimportantindendrites, certaincommonly-usedCl- channelblockerswere wheretheyareinvolvedinintegratingsynapticinputs moderatelygoodblockersofKv4.3andKv4.2 andregulatingbackpropagationofactionpotentials. channelsinexpressionsystems.Niflumicacid,at100 Thesediscoverieshavepromptedagreatdealof µM,causesaprofoundreductionofKv4.3channels interestinthesechannelsubunits,especiallyfrom expressedinoocytes,accompaniedbyan~-10mV pharmaceuticalquarters. shiftinsteadystateactivationandinactivationcurves. TheCl- channelblockerDIDS(100µM)inhibitsKv4.3 ThebiophysicalpropertiesofKv4channelsin current,producingadramatic(~3.3-fold)slowingof expressionsystemsandIto intheheartarevery recoveryfrominactivation,butwithoutmajorchanges similar;bothactivatebeyondabout-40mV,andare inactivationandinactivationcurves.Kv4.2channels, rapidlyinactivating,withhalf-decaytimesof50msor despitehavingahighaminoacididentitytoKv4.3 lessatroomtemperature.Kv4channelsoften channels(~75%)weremuchlesssensitivetotheCl- inactivatewithamulti-exponentialdecay(inactivation channelblockers.Interestingly,Wangetal (2000)95 gatingisverycomplexseee.g.Bähring etal reportthatnicotineblocksKv4.2andKv4.3currents 86 (2001)),inavoltage-dependentmanner.Single inoocyteswithanIC50 of~40nM,aconcentration channelconductancehasbeenreportedtobeless seenwhensmoking. than10pS(Coetzeeetal (1999)1).Onehallmark,at leastsometimesusefulineliminatingotherKv ItisimportanttonotethatKv4subfamilychannelsare subtypes(e.g.Kv3),istherelativeresistanceofthese alsostronglyinfluencedbyaccessoryproteins.The KvchannelsubunitstomMconcentrationsofTEA bestcharacterizedofthesearetheintracellular (seePaketal(1991)87,Dixonetal (1996)88,Coetzee KChIPs(Kv4channelinteractingproteins),whichco- etal (1999)1).However,theyshowmoderate(few localizeandco-immunoprecipitatewithKv4channels mM,e.g.Fisetetal (1997)89)sensitivityto4-AP.For (Anetal (2000)96).Whenco-expressed,Kv4and 39 example,Tseng(1999)hascalculatedanIC50 of KChIPsalterthegating(shiftingactivationtomore 1mMforratKv4.2expressedinoocytes.Although,as negativepotentials)andamplitudeoftheα-subunits, discussedabove,IC50 values,especiallyfora thechannelgatingeffectsprobablyarisingfromthe complicatedblockerlike4-AP,willdepend abilityoftheseaccessoryproteinstobindintracellular considerablyonpulseprotocolsusedand Ca2+.Severalotherintracellularproteinshavealso preparationsemployed,soitisbettertousetherange recentlybeenshowntointeractwithKv4channels, here,ratherthandefinitivevalues. includingKvββ1andKv2subunits(seeabove,Yang etal (2001)97),theMinKrelatedpeptide1(MiRP1, Onceagain,naturehasprovidedwhatareprobably Zhangetal (2001a)98)andthenewestaddition,asyet themostselectivetoolsfortheidentificationofKv4- unidentified,calledKAF(Kv4channelaccelerating containingionchannels.Sanguinettietal (1997)90 factor,Nadaletal (2001)99).Pharmacologicaldataon identifiedthreepeptidictoxins(heteropodatoxins, theeffectsoftheseco-associationsincellsarenot HpTX),isolatedfromthevenomofMalaysian yetavailable,butbyalteringkinetics,andthreshold Heteropodavenatoria spiders,whichintheheart etc,itwouldnotbesurprisingifchangesindrug prolongventricularactionpotentialsbyblockingI.to sensitivitywereseen.Indeed,MiRP1retards4-AP HpTX1-3areabout30aminoacidslong,andshare unbindingfromKv4.2channels(Zhang etal about40%sequenceidentity.Six cysteineresidues (2001a)98),andmodulationofKv4channelsubunits withinthesequencewouldindicatetightdisulphide byarachidonicacidisdependentonKChIPS bondinginthetertiarystructure,perhapsmaking (Holmqvistetal (2001)100). modellinganddeterminationoftheiractivesite(s) easier(seeBernardetal (2000)91).HpTX3isquite EAGchannels similar(~39%)totheChileantarantulatoxin, hanatoxin2,whichblocksKv2.1potassiumchannels TheoverallstructuralsimilaritybetweenEAG(ether- + (seeabove,SwartzandMacKinnon(1995)67).In à-go-go )andtheothervoltagegatedKchannels Xenopus oocytes,allthreeHpTX’sblockKv4.2ina discussedaboveisquitelow,exceptforthepore voltage-dependentmanner,andslowtheactivation region(indeed,theyareclosertothecyclicnucleotide andinactivationratesofthesechannels.Sanguinetti gatedchannels).EAGcomprisestheeag,erg(eag- + etal (1997)90 showedthatHpTX’salsoshiftsteady relatedgenes)andelk(eag-likeKchannels) stateinactivation,andstrongdepolarizationsrelieve subfamilies.eag1andeag2;erg1-3,andelk2and3 heteropodatoxinblock.Inaddition,theseauthors havebeenidentifiedthusfarinthemammalianbrain 101 reportedthatKv4.2channelswerealsoblockedby (Saganichetal (2001)),althoughrolesformanyof hanatoxin(73%by500nMat0mV). theseinthebrainhaveyettobediscovered.

Twootherusefultoxinshavebeenisolatedfrom HERG(humanether-à-go-go related)isoneofthe tarantulaspiders,whicharealsoselectiveforKv4.2 mostnotablemembersoftheEAGfamilyofvoltage- + andKv4.3channels.Diochotetal (1999)92 reported gatedKchannelsinmammals.TheHERGchannelis thatphrixotoxins(29-31aminoacidslong,fromthe widelyheldtobethemajorsubunitofthecardiac tarantulaPhrixotrichusauratus),blockKv4.3and ‘rapiddelayedrectifier’,Ikr,intheheart(Sanguinetti et 102103 Kv4.2intherange5-70nM,shiftingtheactivation al(1995),Trudeauetal(1995),Curran etal 104 curveinthepositivedirection,andagain,blockcan (1995)).DefectsintheHERGgenehavebeen berelievedbystrongdepolarizations.Thesetoxins showntounderlieLQT2syndrome(producedbya have~50%sequenceidentitywithheteropodatoxins, varietyofmechanisms,includingabnormalchannel and20%identitywiththehanatoxins. processing,‘nonfunctional’andgating-shifted

7 channels)andHERGhasarousedconsiderable actionpotential.Theauthorsalsoconcludethatblock interestasadrugtarget.HERGcurrentshaveafairly ofHERGchannelsoccursviatheclosed-channel highthresholdforactivation(above-50mV,V1/2 ~-13 route.Thisstudyprovidesaninterestingand mV),slowactivation,andveryfastinactivation,which importantexampleofhowcombinationsofdrugscan producesinwardrectificationatpositivepotentials leadtomoreseriouseffectsthanonealone. (Sanguinettietal (1995)102).Co-associationofHERG withMiRP1subunitsisrequiredtofullyreproduceIkr Cisapride(Propulsid)isacommonly-used currents.Theoligomericcomplexhasaslight gastrointestinalprokineticagentusedinpatients rightwardshiftinactivation,changedsinglechannel sufferingfromreflux.However,thereareindications conductance,andaccelerateddeactivation. ofpatientsacquiringQTsyndromeathighdosesof Importantly,thereareanumberofhumanmutations cisapride(seeRampeetal (1997)111).Rampe etal inMiRP1,whichleadtoarrhythmias. (1997)111 showedthattheseunwantedcardiacside effectsmightbeassociatedwithblockofHERGby HERGchannelsareselectivelyblockedbyclassIII cisapride,asithasanIC50 forblockofHERGaslow antiarrhythmicmethanesulphonamidedrugs,suchas as45nM. dofetilide,E-4031andMK-499.E-4031hasaQT waveprolongingeffect,andisthoughttoblockIkr TheClassIIIantiarrhythmicagentazimilideblocksa selectively.Intheoriginalreports,E-4031was numberofvoltage-gatedchannelsintheheart, 115 reportedtoblockIkr inguineapigmyocyteswithan includingHERG.Buschetal (1998)haveshown IC50 of~400nM(SanguinettiandJurkiewicz thatazimilideblocksHERGchannelsinareverse-use (1990)105),althoughrecentexperimentshavelowered dependentandvoltageindependentmanner,in thisIC50valueforIkr considerably,toaround10nM contrasttoalloftheothercommonHERGchannel (Liuetal (1996)106).Similarly,therearelarge blockers(E-4031,dofetelide,terfenadine,astemizol, differencesinIC50 blockofHERGindifferent clofiliumandhaloperidol),whichshowbothpositive expressionsystems(oocytes:590nM,Trudeau etal use-dependenceandvoltage-dependenceintheir (1995)103,HEKcells~8nM,Zhouetal (1998)107).Itis blockade.Thismeansthattheblockandapparent notknownwhetherthesedifferencesmaybe affinityofazimilidedecreasedwithHERGchannel explainedbyvariationsinexperimentaltechnique,or activationfrequency(e.g.IC50’sof1.4µMat0.1Hz, preparation. 5.2µMat1Hz).

Herzbergetal (1998)108 ‘transferred’keyregionsof Haloperidolisabutyrophenoeantipsychoticdrug, HERGandanother,structurallyrelatedmemberofthe usedinthetreatmentofschizophrenia,butitswide Eagfamily,M-eag,makingnovelchimerasthathave useiscomplicatedbyseveralcasesofacquiredQT toldusmuchabouttheregionsinvolvedinfast syndrome.Centrally,itactsbyblockingdopamine inactivation,aswellassensitivitytoE-4031.M-eag receptors,butSuessbrichetal (1997b)116 showed channelsaresubstantiallyinsensitivetothe thatitcanalsoeffectivelyblock(IC50 ~1µM)HERG antiarrhythmic,butthechimericchannel,havingtheP channelsinoocytes.Haloperidolpreferentiallyblocks regionofHERGandhalfitsS6segmentina HERGchannelsintheinactivatedstate,supportedby backgroundofM-eag,hadenoughofthedrugbinding afourfoldreducedblockinaHERGmutant(S631A), sitetogivethesamesensitivitytoE-4031(~350nM whichhasmarkedlyreducedinactivation(Suessbrich each). etal (1997b)116).

TheClassIIIantiarrhythmicclofilium(aquaternary Rampeetal (1998)117 have shownthatthe ammoniumderivative),blocksanumberofdelayed antipsychoticagentsertindoleblocksHERGcurrents rectifierK+ currentsintheheart,butis‘selective’for inmammaliancelllinesatnMaffinity.(Thismaybe HERGcurrentsatconcentrationsseveralordersof relatedtoreportsofacquiredlongQTsyndromein magnitudelower,suggestingthatHERGisthemain patientsbeingtreatedwiththisantipsychotic).HERG locusofactionintheheart(Suessbrich etal currentsareincreasedbyincreasesinextracellular 109 (1997a)).TheIC50 forclofiliumis~150nMat+40 potassium,despiteadecreaseindrivingforce mV,and~250nMat0mV,whilstatertiaryanalogue (Sanguinettietal (1995)102,Schonherrand ofthisdrug,LY97241,was10timesmorepotentstill. Heinemann(1996)118);asimilareffectisalsoseenfor 105 Dofetilide,anothermethanesulfonamide,alsoblocks Ikr inheart(SanguinettiandJurkeiwicz(1990)). + HERG.EstimatesofIC50 varyfrom~12-15nMin Increasesin[K]o decreasetheeffectivenessofthe mammaliancelllines(SnydersandChaudhary HERGblockersclofilium(Suessbrichetal (1997b))116 (1996)110,Rampeetal (1997)111)to35nMinoocyte andazimilide(Buschetal (1998))115 anddofetilideis + patches,and~600nMin‘whole’oocytes(Kiehn etal ~26timeslesseffectiveagainstIkr as[K]o increases (1996)112).Again,onehastobecautiouswhen fromonly1-8mM(YangandRoden(1996)119).Itis + comparingresultsobtainedwithdifferentexpression likelythatthe[K]o effectislinkedtomodulationofC- systems,andtechniques. typeinactivation.Anotherroutetomodificationof HERGisavailable,asKiehn(2000)120 showedthat Terfenadine(Seldane),isanonsedating activationofproteinkinaseAleadstoasuppression antihistamine,whoseusewasfoundtoleadto ofHERGcurrents,duetoarightwardshiftinthe prolongedQTsyndrome(seee.g.Roy etal steady-stateactivationcurve(whichcanbeasgreat (1996)113).ThisdrugblocksHERGinHEKcellswith as+35mV).Itwillbeofinteresttolearnunderwhat 111 anIC50 of56nM(Rampeetal (1997)). conditionsthisprevailsinvivo,sinceanythingwhich modifiestheavailabilityofachannel,willalso Interestingly,whenterfenadineisusedintreatmentin contributetoitsdrugresponsiveness. commonwiththefungicideketoconazole(thelatter usedforathletesfootordandruff),patients Halofantrineisanextensivelyusedantimalarial, occasionallypresentwithlongQTsymptoms.Both whichhasbeenassociatedwithsomeunfortunate compoundsusethesamecytochromeP450pathway, cardiacsideeffects,oftenleadingtosuddendeath. andthecompetitionbetweenthetworaisesplasma Tieetal (2000)121 haveshownthathalofantrine 114 terfenadinelevels.Dumaineetal (1998)recently blocksHERGtailcurrentswithanIC50 of200nM, showedthatketoconazoleitselfcaninhibitHERG some10-foldlessthanthetherapeuticdosein currents(intheµMrange),andthatco-applicationof patients.Furthermore,halofantrineisoftenco- terfenadineandketoconazoleprolongedthecardiac administeredwithmefloquine,anotherantimalarial,

8 whichalsoblocksHERGchannelsandtheslow SelectiveinhibitorsofIKs anditsmolecularconterpart delayedrectifierK+ channelinheart(Kang etal arethereforeofinterestastargetsforthe (2001)122).Cocainealsocausescardiacarrhythmias developmentofnovelclassIIIantiarrhythmicdrugs. andsuddendeath,andcocaineabusehasbeen Mutationsinthischannelcomplexhavebeenlinked reportedtoresultinQTwaveprolongationinman. tooneoftheformsofinheritedlongQTsyndrome Zhangetal(2001b)123andFerreiraetal (2001)124 (LQT1),leadingtocardiacarrhythmias(Wang etal foundthatcocaineblocksHERGchannelswithan (1996)130). KCNQ1canalsoco-assemblewith IC50 of4-7µM,anditsmetaboliteswereevenmore KCNE3.Thisassociationisshowntogreatlyaffect potent. KCNQ1gating,leadingtoconstituitivelyopen channels.Insituhybridisationandcomparisonwith Recently,Pardo-Lopezetal (2002)125 havereported nativecurrentsindicatethattheKCNQ1/KCNE3 onatoxinderivedfromscorpion,ergtoxin(ErgTx), channelisthemolecularcorrelateofthecAMP- + whichspecificallyblocksHERGchannels,withaKd regulatedKcurrent,providingastandingoutward,or value~11nM. non-inactivatingK+ currentpresentincoloniccrypt cells(Schroederetal (2000b)133). Thistoxindidnotblockeithermouseeagorelk channels,beingabletobindtotheaminoacids Severalusefultoolsarealreadytohandwithregards betweenS5andthePloop. toinhibitionofKCNQ1anditscomplexes.Clofilium (10µµM)inhibitsKCNQ1withanIC50 of<10M(4). Pharmacologyfortheelksubfamilychannelsismuch Linopirdine(IC50 ~9µµM)andXE911(IC50 ~0.8M) morelimited.Rodentelkchannelsareblockedby arealsopotentinhibitorsofKCNQ1(Wang etal externalBa2+ at1mM,butareresistantto100mM (1998)135).Incontrast,theblockersCTX,4-APand TEA,10µME4031and4-AP(Engeland etal E4031havenoeffectonKCNQ1currents(Yang etal (1998)126,Shietal(1996)66,Trudeauetal (1999)127). (1997)131).Interestingly,benzodiazepinederived compoundsareprovingusefulblockersand KCNQchannels modulatorsofKCNQ1.Thenovelbenzodiazepine,L- 735,821,whichincreasestheventricularaction + Arecentlydiscoveredfamilyofvoltage-gatedK potentialduration,blocksKCNQ1channelswithan channelsistheKCNQgenefamily.Todate,5genes ICof80nM(Salataetal (1996)136)butathigher ofthisfamily(KCNQ1-5),allshowntoencodeK+ 50 concentrationsitalsoblocksKCNQ2subunits(IC=50 channelsubunits,havebeenidentified.All5known 1.5µM;Tineletal (1998)137).Incontrast,thenovel KCNQproteinscanformhomomericchannelswith benzodiazepineL-364,373apparentlyfacilitatesthe theformationofheteromersseeminglyrestrictedto openingofKCNQ1asmeasuredbyaleftwardshiftin certaincombinations(seebelow).Ingeneral, theactivationcurveforIKs inguineapigventricular however,expressionofthesechannelsinduces myocytes(Salataetal (1998)138).Morerecently,the slowlyactivating(atvoltagespositiveto-60mV), stereospecificinhibitionofI,viause-dependent + Ks outwardlyrectifying,voltage-dependentKcurrents blockoftheKCNQ1/minKcomplexbythe displayinglittleornoinactivation. enantiomer(-)-[3RS,4]-chromanol293Bhasbeen demonstrated(IC=1.36µM;Yangetal (2000)139). Significantinterestinthesesubunitshasbeen 50 promptedbythediscoverythatmostoftheexpressed KCNQ2subfamilychannels familyofchannelgeneshaveclear,important physiologicalcorrelatesandthatmutationsinthese KCNQ2hasbeensuccessfullyexpressedinarange genescanbeenlinkedtospecifichumandiseases, ofcelltypes.Ingeneral,KCNQ2homomersproduce highlightingtheirpotentialwithrespecttothe slowlyactivatingoutwardcurrentsondepolarising developmentofnovel,clinicallyuseful,drugs.Two stepspositiveto-60mV,showingslightinward excellentreviewsconcerningKCNQchannelsare rectificationatmorepositivepotentials.However,the available(Jentsch(2000)128,Robbins(2001)129). biophysicalparametersseemtodependonthe expressionsystemand/orthevoltageprotocol KCNQ1subfamilychannels (compareBiervertetal(1998)140 withSelyanko etal (2000)141). KCNQ1,previouslynamedKvLQT1,wasthefirstof theKCNQfamilytobeidentifiedandisexpressed KCNQ2subunitsarewidelydistributedinthebrain stronglyinhumanheart,cochlea,kidney,placenta, 130 andhavebeenshowntocometogetherwithKCNQ3 lung,colonandpancreas(Wangetal (1996),Yang inheteromultimerstoyieldlargercurrentswithslightly etal (1997)131).HomomericKCNQ1channels 132 changed gatingkineticsandsensitivitytoinhibitors expressedinoocytes(Barhaninetal (1996), (Wangetal(1998)135,Hadleyetal (2000)142).Itis Schroederetal (2000b)133)andmammaliancelllines 134131 nowgenerallyacceptedthatthischannelcomplex (Sanguinettietal(1996),Yangetal (1997)) formsthemolecularcorrelateofthe‘M-current’,one exhibitdelayedrectifiercurrentswhichactivateon ofthekeyregulatorsofmembraneexcitabilityinmany depolarisationwithaV1/2 of~-10to-20mV,although neurones(Wangetal (1998)135).Thisfindinghas activationkineticsdovarybetweenexpression provokedfurther,intenseinterestinthepharmacology systems. andregulationofKCNQsubunits(seeJentsch (2000)128,Robbins(2001)129). However,itsco-assemblywithmembersoftheKCNE genefamilyappearstoyieldchannelcomplexesof PharmacologicalstudiesthusfarrevealthatKCNQ2 greatestfunctionalimportance.Forexample,inthe isresistantto2mM4-AP(Yangetal (1998)143),but heart,KCNQ1isassociatedwiththeproductofthe blockedbyTEAwithanICof160µM(Wang etal KCNE1gene,theminKchannelprotein(asmall130 50 (1998)135),duetothepresenceofatyrosineresidue aminoacidwithasingletransmembranedomain),an intheporeregion.LinopirdineblocksKCNQ2withan interactionwhichenhancescurrentamplitudeand ICof~4µM,andXE991blockisaboutequipotent slowsactivation.Thischannelcomplexhasbeen 50 withthatonKCNQ1channels.E4031,theselective showntounderlietheslowcomponentofthecardiac inhibitoroftheHERGchanneldoesnotblockKCNQ2 delayedrectifier,I,whichisinvolvedin Ks at10µM.Similarly,neitherclofiliumnorCTXhavean repolarizationoftheventricularactionpotential effectonKCNQ2currents(Yangetal (1998)143).The (Sanguinettietal(1996)134,Barhaninetal (1996)132). enhancedcurrentexhibitedbytheheteromultimeric

9 KCNQ2/KCNQ3channelshowssimilarsensitivityto distributionofKNCQ4inthebrain mayrulethisoutas linopirdinecomparedwiththeindividualhomomers amajorcontributortoM-currentsinmanyneurones. (IC50 ~4µM)andappearstoretainthesensitivityof theKCNQ2homomertoXE991.Reportsregarding KCNQ5subfamilychannels blockbyTEAvaryslightly,butindicatethatthe sensitivityoftheKCNQ2channelisretained(Wang et ThemostrecentlydiscoveredoftheKCNQgenes, al(1998)135,Hadleyetal (2000)142).Retigabinehas KCNQ5isbroadlyexpressedinthebrainandhas beenshowntoactasan‘agonist’ofhomomericand beenshowntobepresentinsuperiorcervicalganglia heteromultimericcombinationsofKCNQ2and andNG108-15cells.Itshouldalsobenotedthat KCNQ3channels,byshiftingtheactivationcurveto mRNAforKCNQ5hasbeendetectedinskeletal theleft(Tatulianetal (2001)144). muscle,althoughitsfunctionthereisasyetunclear (Schroederetal(2000a)147,Lercheetal (2000)149). MutationsinKCNQ2(andKCNQ3)areassociated LikeotherKCNQsubunits,KCNQ5homomeric withaconditioninhumaninfantscalledbenign channelsyieldcurrentsthatactivateslowlyon familialneonatalconvulsions(BNFC).Typically, depolarisation.Someinwardrectificationisseenat verypositivepotentials(Schroederetal (2000a),147 convulsiveepisodesstartshortlyafterbirthand 149 usuallysubsidewithinthreemonths(Biervert etal Lercheetal (2000) ). (1998)140).SelectiveM-channelopenersmay thereforehavepotentialaseffectiveantiepileptics. Recentstudiesusingdominantnegativemutantsand mutantswithdifferentsensitivitiestoinhibitorshave KCNQ3subfamilychannels suggestedthatKCNQ5canformheteromeric channelswithKCNQ3butprobablynotwith1,2,or4. ExpressionofhomomericKCNQ3channelsalonehas Co-expressionwithKCNQ3slightlychangedits beenweak,atmost.Wherechannelshave kineticpropertiesandincreasedcurrentamplitude. expressed,currentsactivateatpotentialspositiveto Additionally,KCNQ5doesnotseemtofunctionally 60mV,withaV1/2 of~-37mV.Currentsalsoappear interactwiththeKCNEfamily(Schroeder etal tomarkedlyrectifyinwardlyatmorepostivepotentials (2000a)147,Lercheetal (2000)149). (Yangetal(1998)143,Selyankoetal (2000)141).Like KCNQ2,thishomomericchannelissensitive to Intermsofpharmacology,KCNQ5currentsshowlittle linopirdine(IC50 ~4µM)and,toalesserextent,to sensitivitytoTEA(IC50 >30mM).Thechannelis XE991.However,unlikeKCNQ2,KCNQ3isresistant weaklysensitivetolinopirdinewithreportedIC50’s toexternalTEAat5mM,andisblocked(about30%) rangingfrom16-51µM(Lercheetal (2000),149 by10µMclofilium.CTX,4-APandE4031arewithout Schroederetal (2000a)147).KCNQ5alsobeen effect(Yangetal (1998)143). demonstratedtoshowsomesensitivitytoXE991with 95 anIC50 of~65µM(Wangetal (2000)).Withrespect KCNQ3ismostwellstudiedwheninheteromultimeric tochannelactivators,enhancementofbothKCNQ5 assemblywithKCNQ2,followingitsidentificationas andKCNQ3+KCNQ5channelcurrentsbyretigabine themolecularcorrelateoftheM-current(Wang etal hasbeenreported(Shroederetal (2000),147 (1998)135,seeabove).Itcan,however,alsoform Wickendenetal (2001)150). functionalheteromultimerswithKCNQ4andKCNQ5 (seebelow). ThestudiesofSchroederetalandLerche etal (2000)147,149 havesuggestedthattheformationof KCNQ4subfamilychannels functionalheteromersbyKCNQ5withKCNQ3inthe CNSandperipheralgangliamaycontributefurtherto KCNQ4displaysamuchmorerestrictedexpression Mchanneldiversityinnativetissue.Accordingly, patternthantheotherKCNQchannels,being giventhattheexpressionpatternofKCNQ5issimilar localisedtothe sensoryhaircellsintheinnerearand to2and3andthatitcanformheteromerswith insectionsofthecentralauditorypathway(Kubisch et KCNQ3,itcouldbespeculatedthatKCNQ5 145146 al(1999)),Kharkovetsetal (2000)).Itforms mutationsmayalsocauseepilepsy.Todate,however, homomericpotassiumchannelswhichactivateslowly nospecificpathologyhasbeenlinkedtoKCNQ5 ondepolarisationanddisplayaV1/2 of~-10mVin channeldefects. oocytesand-20mVinCHOcells(Kubisch etal 145141 (1999),Selyankoetal (2000)).Inaddition, Conclusion studiesinoocyteshaveshownthatwhileKCNQ4 doesnotfunctionallyinteractwithKCNQ1or2, Wehavediscussedonlyafractionofthehuge coexpressionwithKCNQ3yieldsacurrentamplitude numberofagentsthatactatvoltage-gatedK+ greaterthanthehomomultimericsum,suggestingit channels.Thebest,andmostusefulofthesearethe canformheteromerswithKCNQ3(Kubisch etal naturally-occurringpeptidicblockers.However,westill (1999)145).Atpresent,thereislittlepharmacological donothavehighaffinityselectiveblockersformost dataavailableforthesechannels;KCNQ4showsan channels,andthisisachallengeforpharmacologists intermediatesensitivitytoTEAwhencomparedtothe andmedicinalchemists.Unfortunately,thereareeven + otherKCNQchannels,withanIC50 of3mM(Hadley fewerenhancers(oragonists)forvoltage-gatedK etal(2000)142,Schroederetal (2000a)147).Incontrast channels;suchagentswouldbetremendouslyuseful toalloftheotherKCNQsubunits,itisfairlyresistant inincreasingthebrakingpowerofK+ currentsin tolinopirdine(IC50 >200µM). mammaliancells.Nevertheless,thefuturelooks bright,especiallysincethereissuchintenseeffortin Withregardtochannelopeners,retigabinehasbeen thefieldofK+ channels. demonstratedtoproduceahyperpolarizingshiftofthe activationcurvesofKCNQ4channelsexpressedin Acknowledgements CHOandHEK293cells(Tatulianetal (2001),144 Schroderetal (2001)148). BRwouldliketothankallthemembersofhislab, pastandpresent,fortheirtime,efforts,andexpertise. MutationsinKCNQ4,perhapsnotsurprisinglyfromits distribution,havebeenassociatedwith aformof inheriteddeafness(Jentsch(2000)128).Recentdata alsosuggeststhatKCNQ4maycontributetoM- channeldiversity.However,theratherrestricted

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11 POTASSIUMCHANNELMODULATORSAVAILABLEFROMTOCRIS

ATP-Activated 1377Cromakalim...... KATP channelopener + 0964Diazoxide...... Kchannelopener(K)ATP + 0911Glibenclamide...... Kchannelblocker(K)ATP 1378Levcromakalim...... KATP channelopener.Activeenantiomerof(1377) + 0583Minoxidil...... Kchannel(KATP)opener 1355P1075...... PotentKATP channelopener + 1503Pinacidil...... Kchannelopener.ActivatesKATP channels 0882ZM226600...... KATP channelopener

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Voltage-Gated + 0876AM92016...... Kchannelblocker(K)V 1412Chromanol293B...... IKsblocker.AlsoblocksICFTR 1475(-)-[3RS,4]-Chromanol293B...... IKs blocker.Enantiomerof(1412) 1399CP339818...... Non-peptide,potentKv1.3channelblocker

Other 0385N-[2-(Acetoxy)ethyl]-3-...... K+ channelopener 09404-Aminopyridine...... K+ channelblocker pyridinecarboxamide 0416YS-035...... InhibitsK+ outward/pacemakercurrent Localregulationsmayrestrictthesaleofsomeoftheaboveproductsincertainterritories.PleaseconsultyourlocalTocrisCooksonofficeor distributorforfurtherdetails.

PharmacologyofVoltage-GatedK+ Channels, TocrisReviews No.22,December2002

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