BrianFKingandAndreaTownsend- P2XReceptors Nicholson P2Xreceptors(P2XRs)areligand-gatedion-channels (LGICs)widelydistributedamongstvarioustissue UniversityCollegeLondon,GowerStreet, types,includingautonomic,central,entericand London,WC1E6BT,UK. sensoryneurons,cochlearandretinalcells, endotheliumandepithelium,vascularandvisceral BrianKingisaSeniorLecturerinthe smoothmuscle,heartanddevelopingskeletal DepartmentofPhysiology,andAndrea muscle,bone,haemopoieticcells.TheseLGICsserve Townsend-NicholsonaLecturerinthe tocontroltheexcitabilityoftheirhostcellsthrough DepartmentofBiochemistryandMolecular twoprocesses:i)influxofextracellularsodiumionsto elicitdepolarisation,andii)influxofextracellular Biology.Theyshareacommoninterestin calciumionstoactivateinternalenzymes. purinesignallingatrecombinantandnative P1andP2receptorsandhaveworked P2XRassembly togetheroverthelastsevenyearsonthe Sevenbuildingblocks, orsubunitproteins,are isolationandcharacterisationofnucleotide involvedintheconstructionofP2XRs.Theseseven receptors. buildingblocks–theP2X17toP2Xsubunits–have beenclonedfromhumanandratcDNAlibrariesand, insomecases,alsofrommouse,guinea-pig,chick andzebrafishlibraries.1,2 Fewstructuraldifferences havebeennotedbetweenspeciesorthologuesofthe Introduction sameP2Xsubunit.However,thedegreeofhomology canbeaslittleas27%betweendifferentsubunitsof Extracellularnucleotidesignallingmolecules,suchas thesamespecies.1 Furthermore,theP2Xgenesare ATP,ADP,UTPandUDP,workthroughspecific complexand,duringtranscription,canundergo ionotropic(P2X)ormetabotropic(P2Y)cell-surface alternativesplicingwhich,subsequently,generates receptors.Theuseofanyligandasasignalling variantsubunitproteinsthatareeithernon-functional, moleculenecessitatesamechanismforits dominantnegativeormodulatory. inactivationandso,purinenucleotidesarehydrolysed toadenosine,anucleosidethatactsthrough Eachofthesubunitproteinscanformhomomeric metabotropicadenosine(P1)receptorsubtypes,to P2XRassemblies,althoughthereappeartobe exertphysiologicaleffectsinalmostallorgan difficultiesformingP2Xreceptors3 andP2X systems.Inosine,formedbythedeaminationof 56 receptors.4 Forsuchdifficulties,post-translational adenosine,hasalsobeenshowntohaveagonist modificationofsubunitproteins–particularly activityatnucleosidereceptors.Furthermore, glycosylation–hasastronginfluenceonthe dinucleotidesaresignallingmoleculesintheirown functionalityofmembrane-traffickedP2XRs.5,6,7 Itis right–notonlyatP2receptors,butalsoatspecific currentlybelievedthateitherthreeorsixglycosylated ApAreceptors–and,throughdegradation,can n subunitsmakeafunctionalP2XR,byformingeithera generatenucleosidesandnucleotidestobroaden stretchedtrimerorahexamerofconjoinedtrimers.2,8 theirextracellulartargets. Inadditiontohomomericassembliesofidentical subunits,P2Xproteinsalsoformheteromeric SevenP2X,eightP2Yandfouradenosinereceptor assembliesbyusingtwoorthreebuildingblocks. proteinshavebeenclonedandcharacterised.Despite WheretwoP2Xsubunitsareinvolved, theubiquityofbothpurinergicligandsand immunoprecipitationstudieshavepredictedupto11 purinoceptors,thedifferentialexpressionof heteromericreceptorsubtypes.9 Here,aninteresting purinoceptorsubtypesandligand-metabolising patternhasemerged–P2Xsubunitsaremost enzymesallowsforthegenerationofveryspecific 7 discerningandonlywillformhomomericreceptors, physiologicalresponsesinparticularcelltypesor whereasP2Xsubunitsaremostpromiscuousand tissues.Itisforthisreasonthatnucleotideand 5 willheteropolymerizewithallP2Xsubunits(except nucleosidereceptorshavenowbecometherapeutic P2X).OtherP2Xsubunitscanpolymerisewithsome, targetsforthetreatmentofconditionsasdiverseas 7 butnotall,P2Xsubunits.Asyet,noonehas pain,urinaryincontinence,strokeanddepression, undertakenasystematicstudyofheteromeric andthefieldofpurinoceptorresearchhasexpanded assembliesinvolvingthreedifferentsubunits, significantlyinthelasttenyears.Inthisbriefreview, althoughsuchassembliesmustsurelyoccurinnative weexamineanumberofthepharmacologicaltools tissues,sinceanumberoftissues(e.g.dorsalroot currentlyavailableforthestudyofnucleotideand ganglia,autonomicgangliaandsmoothmusclecells) nucleosidereceptors. possessinabundancetranscriptsandproteinsfor severalP2Xsubunits. TocrisCooksonLtd.,UK TocrisCooksonInc.,USA Tel:+44(0)1179826551 Tel:(800)421-3701 Fax:+44(0)1179826552 Fax:(800)483-1993 [email protected] [email protected] [email protected] www.tocris.com [email protected] Figure1.Thepurinoceptorfamilyofcell-surfacereceptors Thepurinoceptorfamily,atthispointintime,comprises5structurallyandpharmacologicallydistinctgroups–the adeninereceptorgroup(AdenR),adenosinereceptors(ARs),metabotropicnucleotidereceptors(P2YRs), ionotropicnucleotidereceptors(P2XRs)intheirhomomericandheteromericforms,anddinucleotidereceptors (ApnnAR).Thelastgroup(ApARs)mighteventuallybesubdividedintoionotropicandmetabotropicsubgroups,for whichthereispharmacological,butnotyetstructural,evidence.Finally,membersoftheARandP2YRgroups appeartoheterodimerise,althoughonlytheA11/Ycomplexhasbeenstudiedfunctionally. The“purinergic”family AdenRARsP2YRsP2XRsApnARs 1R A1AAA2A2B 3 ? A11/Y YYYYY124611Y12YY1314 X1XXXXXX234567 XXXXX1/22/32/61/54/6 Todate,sevenhomomericP2XRs(P2X17toP2X)and itself.NeitherADPnorAMP(whenpurifiedof fiveheteromericassemblies(P2X1/2,P2X1/5,P2X,2/3 contaminatingATP)willactivateP2Xreceptors, P2X2/6andP2X4/6)havebeencharacterisedbytheir exceptathomomericassembliesofP2X1 splice biophysicalandpharmacologicalproperties. variants(exon6deleted)whichareactivatedbyADP 14 OperationaldifferencesbetweenmanyoftheseP2XR andnotATP,andathomomericP2X7 receptors assembliesaresurprisinglysubtle.Also,mosttissues whichwillrespondtobothADPandAMP(withina expressbothhomomericandheteromericP2XRsofa periodoftime)afterbeingactivatedfirstbyATP.15 The similarpharmacologicalnature.Asaresult,itisan naturally-occurringdiadenosinepolyphosphates inordinatelydifficultprocesstodeterminetheidentity (ApnA,n=3-7)andclosely-relateddinucleotides ofnativeP2XRs.Atthispointintime,itisinadvisable (ApnG,n=3-6)alsoactivateP2XRs,butnonewith tocategorisenativeP2XRsasP2X,123P2X,P2X,etc. eitherthepotencyorefficacyofATP.Membersofthe and,instead,theyshouldbeassociatedwitha diadenosinepolyphosphatefamilyaresoclosein particularphenotype(e.g.P2X123-like,P2X-like,P2X- potencyandefficacyatP2Xassembliesthatthese like,etc.).TherolesofseveralP2Xreceptorsubunits substancescannotbeusedreliably(andalone)as innativechannelshavebeenexaminedbythe discriminatorsofP2Xsubtypes.Thediphosphates, generationofknockoutmice.Nullmutationsforthe Ap22AandApG,arenotagonistsatP2XRs,inkeeping 101112 P2X1,P2X37andP2Xsubunits,and withtherelativeinactivityofADP. 13 overexpressingP2X1 transgenicmice,havebeen described,implicatingP2Xreceptorsinprocessesas AmongstrecombinantP2XRs,thepotencyofATPcan diverseasfertility,thrombosis,painandcytokine varyenormously,withEC50 valuesrangingfrom50 production.invivo nMto300mM,dependingonsubunit composition.1,2,16-20 Althoughmutationalanalyses P2XRagonism havebeencarriedout,astructuralbasishasnotbeen AlloftheknownP2Xassembliesareactivatedby clearlyestablishedforthisobservedvariabilityin ATP,whichisthemostpotentofthenaturally- agonistpotency.AcommonATPrecognitionsitehas 2122 occurringnucleosidetriphosphates(NTPs).Other beenmappedforP2X12andP2Xsubunits,and NTPscanworkatcertainreceptorsubtypes(e.g.CTP thissiteissharedbytheremainingP2Xsubunits. atrecombinantP2X,1P2X4andP2X,55GTPatP2X, However,othermodulatoryfactorsmustclearlycome andUTPatP2X3),butnoneareaspotentasATP intoconsideration.Forinstance,agonistpotencyis 2 Table1.KeyligandsforhomomericP2Xreceptors SubtypeAgonistsAntagonistsPotentiatorsInhibitorsReferences (pEC50)(pIC50)(pEC50/pKa)(pIC50i/pK) + rP2X1 ATP(7.0)NF449(9.5) –H(6.3)1,2,3,17-20, 2-MeSATP(7.0) NF279(7.7) Zn2+ (6.0)25-30,88,89 ATPgS(6.2)NF023(6.7) Gd3+ (6.5) Ap6A(6.0) Suramin(5.7) ApG65(5.7)IpI(8.5) ab,-meATP(5.5)TNP-ATP(9.0) Bz-ATP(4.6)MRS2257(8.3) CTP(4.4)MRS2159(8.0) PPNDS(7.8) PPADS(6.9) RB-2(5.7) (h*)A-317491(4.9) +3+ rP2X2 ATP(5.3)RB-2(6.4)H(7.1)La(5.0)1,2,3,17-20, 2-MeSATP(5.1)NF279(6.4) Zn2+(5.0)Gd3+ (5.0)25-30,90-94 ATPgS(5.1)BBG(5.9)Cu2+(4.8)Ca2+ (1.1) 2+ Ap4A(4.8)TNP-ATP(5.9)Cd(3.5) ab,-meATP(3.0) PPADS(5.8) Suramin(5.0) (h*)A-317491(4.33) 2++ rP2X3 2-MeSATP(6.7) A-317491(7.6)Zn(5.0)H(6.0)1,2,3,17-20, ATP(5.9)NF279(5.8) Ca2+ (1.8)88,89,95,96 ATPgS(5.9)NF449(5.6) (h*)RB-2(5.9) Ap5A(5.5) Suramin(5.4) ApG5 (5.6) NF023(5.0) ab,-meATP(5.7)TNP-ATP(9.5) UTP(4.0)MRS2257(7.7) MRS2159(6.9) PPADS(6.7) IpI5 (5.5) RB-2(4.3) + rP2X4 ATP(5.4)TNP-ATP(4.8)Ivermectin(6.6) H(6.8)1,2,3,17-20, 2-MeSATP(3.6) BBG(3.9)Cibacronblue (5.5)25-30,97-103 CTP(3.5) Propofol(4.3) (h*)BBG(5.5)Zn2+ (5.9) partialagonist:(h*)PPADS(4.6) Cd2+ (5.0) Ap4A(5.5) ab,-meATP(4.2) 2++ rP2X5 ATP(6.4)PPADS(6.7) Zn(4.4)H(5.5)1,2,3,17-20 2-MeSATP(6.4) TNP-ATP(6.3)Ca2+ (3.0) ATPgS(6.5) Suramin(5.8) Bz-ATP(5.9)RB-2(4.7) ab,-meATP(6.0) Ap4A(6.6) GTP(4.6) CTP(4.3) rP2X6 ATP(6.2)–––7 + rP2X7 BzATP(5.2)BBG(8.0)H(6.1)1,2,3,15, 2-MeSATP(5.0)PPADS(4.3) Cu2+ (6.5)17-20,31-33, ATP(3.4)TNP-ATP(~4.3)Zn2+ (4.9)104 Mg2+ (3.3) (mh*)ADP(2.7)(*)KN-62(7.4) Ca2+ (2.5) (mh*)AMP(2.3)(*)KN-04(~6.0)Halideions (BoldtextdenotescompoundsavailablefromTocris) Potencyindicesforagonists,etc.givenas-log10EC50(pEC50)values.Dataforhuman(hm*)andmouse(*) isoformsaregivenforsomekeyligands. 3 stronglyaffectedinonewayoranotherby P2X47andP2X,showtime-dependentchangesin extracellularH+ions(pH)andextracellularZn2+ ions channelpermeabilityandshiftsinreversal 23,24 atalltheknownP2XRsubtypes.Otherextracellular potential.ThiseffectismostprofoundforP2X7 ions,includingNa+,Ba2+,Ca2+,Cd2+,Cu2+,Mg,2+