J. Biol. Chem.-2015-Wang-13907-18

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  AnAccessoryAgonistBindingSitePromotesActivationof   4  2*NicotinicAcetylcholineReceptors * Receivedforpublication,January28,2015,andinrevisedform,June3,2015  Published,JBCPapersinPress,April13,2015,DOI10.1074/jbc.M115.646786 JingyiWang ‡ ,AlexanderKuryatov ‡ ,AaratiSriram ‡ ,ZhuangJin § ,TheodoreM.Kamenecka § ,PaulJ.Kenny ¶ ,andJonLindstrom ‡1 Fromthe ‡ DepartmentofNeuroscience,PerelmanSchoolofMedicineattheUniversityofPennsylvania,Philadelphia,Pennsylvania19104, § DepartmentofMolecularTherapeuticsattheScrippsResearchInstitute,Scripps,Florida33458,and  ¶ DepartmentofPharmacologyandSystemsTherapeutics,IcahnSchoolofMedicineatMountSinai,New York, New York 10029 Background: In(  4  2) 2  4nicotinicacetylcholinereceptors,thereisanagonistbindingsiteatthe  4/  4subunitinterface. Results:  2,  3, and  6 accessory subunits can form an agonist site with  4. These promote activation upon agonist binding at  4/  2 agonist sites. Conclusion:  Accessory subunit agonist sites greatly influence receptor function. Significance:  These sites are promising drug targets. Neuronalnicotinicacetylcholinereceptorscontaining   4,  2,and sometimes other subunits (  4  2* nAChRs) regulate addic-tive and other behavioral effects of nicotine. These nAChRsexist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of   4 and  2subunits and a fifth accessory subunit. A third low affinity AChbindingsiteisformedwhenthisaccessorysubunitis  4butnotif it is  2. Agonists selective for the accessory ACh site, suchas 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283),cannot alone activate a nAChR but can facilitate more efficientactivation in combination with agonists at the canonical   4  2sites. We therefore suggest categorizing agonists according totheirsiteselectivity.NS9283bindstotheaccessoryAChbinding site; thus it is termed an accessory site-selective agonist. Weexpressed (  4  2) 2  concatamers in  Xenopus  oocytes with freeaccessory subunits to obtain defined nAChR stoichiometriesand  4/accessory subunit interfaces. We show that  2,  3,  4,and   6 accessory subunits can form binding sites for ACh andNS9283atinterfaceswith  4subunits,but  2and  4accessory subunits cannot. To permit selective blockage of the accessory site,  4threonine126locatedontheminussideof   4thatcon-tributes to the accessory site, but not the   4  2 sites, wasmutated to cysteine. Alkylation of this cysteine with a thioreac-tivereagentblockedactivityofAChandNS9283attheaccessory site.Accessoryagonistbindingsitesarepromisingdrugtargets. nAChRs 2 contain five homologous subunits organized toform a central cation channel whose opening is gated by thebinding of ACh. Homomeric  7 nAChRs have five  7 subunitsand five ACh binding sites at their extracellular interfaces (1).Heteromeric   4  2 nAChRs assemble into (  4  2) 2  4 or(  4  2) 2  2stoichiometrieswiththreeortwoAChbindingsites(2,3).BindingofAChtoonesiteisabletoactivate,inefficiently,both homomeric and heteromeric nAChRs (4, 5). Binding totwositesinan  7nAChRismoreefficient,andbindingtothreeis most efficient for activation. By contrast, binding to four orfive sites in  7 nAChRs promotes desensitization more rapidly thanactivation(6).In  4  2nAChRs,AChcanbindattheinter-face between  4 and  2 subunits (abbreviated as  4/  2) wherethe  4 subunit on the left forms the plus face of an agonist siteand the   2 subunit on the right forms the minus face. In the(  4  2) 2  4 stoichiometry, a third low affinity ACh site is pres-ent at the   4/  4 interface (2, 3). This results in 4-fold largerresponses evoked by ACh of (  4  2) 2  4 stoichiometry than the(  4  2) 2  2 stoichiometry. Because the high affinity   4/  2 ago-nist sites contribute less than 25% of the total response of (  4  2) 2  4 nAChRs, these nAChRs appear to be low affinity if the high affinity component is not clearly resolved (7, 8). Actu-ally,(  4  2) 2  4nAChRscanbeactivatedbylowconcentrationsofagonistsfromtheirintrinsic  4/  2sitestothesameextentasthe (  4  2) 2  2 nAChRs (2).NS9283 is representative of a new class of selective agentstargeting   4  2 nAChRs that have proven useful in aidingnAChR agonists in reducing neuropathic pain and improvingcognition (9–15). It has been termed a positive allosteric mod-ulator (PAM) because it cannot activate nAChRs by itself butenhances  4  2 nAChR activity in response to agonist stimula-tion (9, 10, 12). However, it was recently established thatNS9283 is neither allosteric nor a modulator. It is not allostericbecause NS9283 acts as a selective agonist at the ACh bindingsiteformedatthe  4/  4interface(16,17).NS9283cannotacti- vate through its action on the  4/  4 site alone. In combinationwith agonists at  4/  2 sites, it produces the high probability of channel opening resulting from increased binding site occu-pancy(12,17).BecauseNS9283achievesitseffectbyoccupyingathirdAChsite,justasanyotherfullagonistwouldatthatsite,it is not a modulator. NS9283 does not exceed the maximum *  Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealthGrant DA030929. 1  To whom correspondence should be addressed: Dept. of Neuroscience,PerelmanSchoolofMedicineoftheUniversityofPennsylvania,217Stem-mler Hall, Philadelphia, PA 19104. Tel.: 215-573-3859; Fax: 215-573-3858;E-mail: JSLKK@mail.med.upenn.edu. 2  The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh,acetylcholine; DMSO, dimethyl sulfoxide; MTSEA, (2-aminoethyl)meth-anethiosulfonate; NS9283, 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzoni-trile; PAM, positive allosteric modulator; SSAg, site-selective agonist; EC,effective concentration.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 22, pp. 13907–13918, May 29, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. MAY29,2015ã VOLUME 290ãNUMBER 22  JOURNAL OF BIOLOGICAL CHEMISTRY   13907   a  t   U NI   VE R S I  DADDE  C HI  L E  on N o v e m b  e r  8  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   activation efficiency of (  4  2) 2  4 nAChRs by ACh (18). Asexpected, NS9283 is without effect on (  4  2) 2  2 nAChRs forlack of the  4/  4 binding site (17, 19).In addition to NS9283, other well known   4  2* nAChRligands also show differential actions on  4/  2 and  4/  4 ago-nist sites. The drug sazetidine is a full agonist at  4/  2 primary agonist sites but does not bind to the   4/  4 accessory AChbindingsite(20–22).CytisineactsatbothtypesofAChbindingsites as a partial agonist (16, 20, 22). Dihydro-  -erythroidine isan antagonist at both   4/  2 and   4/  4 ACh binding sites (3).TheseorthostericligandsaredistinguishedfromPAMsthatactatnon-orthostericAChbindingsites,suchastheCterminusof   4 (23), or transmembrane sites on  7 nAChRs (24–26).To avoid misleading nomenclature and provide nomencla-turethatreflectsthemechanismofaction,wesuggestdesignat-ing sazetidine and NS9283 as ACh binding site site-selectiveagonists (SSAgs). There may also be site-selective antagonists.Based on specific site selectivity, SSAgs can be further dividedintotwogroups:accessorySSAgs,suchasNS9283,andprimary SSAgs, such as sazetidine. Here we confirm and extend what isknownaboutaccessorysiteactivationof   4  2*nAChRs(whichcontain  4,  2, and possibly other subunits).Because   4 and   2 subunits can form conventional AChbinding sites, they are referred to as structural subunits. Thefifth subunit of   4  2* nAChRs, which may or may not form anACh site depending on the subunit, is referred to as an acces-sorysubunit.Somesubunitslike  4and  2canfunctionasbothstructural and accessory subunits, whereas others like   5 and  3usuallyfunctiononlyasaccessorysubunits.Hereweinves-tigate the ability of (  4  2) 2 * nAChRs to form ACh andNS9283 binding sites with the accessory subunits  2,  3,  4,  6,  2,and  4.Functionalimpairmentbyblockingaccessory sites suggests that accessory agonist sites exist and that they promote channel activation from the interface between sev-eral    subunits and   4. ExperimentalProcedures Chemicals— 2-Aminoethyl methanethiosulfonate (MTSEA)was purchased from Toronto Research Chemicals Inc. (NorthYork, Ontario, Canada). NS9283 was synthesized as describedpreviously (27). A 10 m M  stock of NS9283 was prepared indimethylsulfoxide.DilutionsofNS9283andMTSEAwerepre-pared daily in testing buffer before use. All other chemicalswere purchased from Sigma-Aldrich unless otherwise noted. cDNAs and cRNAs— Human   3,   4,   6,   2, and   4 werecloned in this laboratory (28–31). The human   2 subunit wasobtained from OriGene Technologies, Inc. (Rockville, MD).The  2sequencewascutoutwiththerestrictionenzymesSmaIandXhoItoshortentheuntranslatedregionandimprovefunc-tionalexpression.The2.0-kbDNAfragmentcodingfor  2wassubcloned into the pSP64 vector for RNA preparation or intopcDNA3.1/Zeo(  ) (Invitrogen) for human cell transfection.Syntheses of concatamers of    2(AGS) 6  4 (abbreviated as  2-  4) and   2(AGS) 6  4(AGS) 12  2(AGS) 6  4 (abbreviated as  2-  4-  2-  4) were described previously (32, 33). Signal pep-tides of    4,   6, and   2 subunits were analyzed by Signal-3L(34). The mature amino acid sequences were used to number  4,  6, and  2 subunits. Mutations in the dimeric concatamerarenumberedastheyareinsinglesubunitsanddisplayedintheupperrightcornerofthesubunitthatcarriesthemutation.Forexample,  2-  4 T126C means that the threonine at the 126 posi-tionofthe  4subunitisreplacedbyacysteine.Mutationswereintroduced using the QuikChange site-directed mutagenesiskit (Stratagene, La Jolla, CA) following the manufacturer’sinstructions.Theaminoacidsmutatedin  4 T126C ,  2 L121C ,and  6 L250S are underlined:   4, VQWTPPAI;   2, IFWLPPAI;   6,SVLLSLTV. All mutations were confirmed by sequencing.After linearization and purification of cDNAs, RNA tran-scripts were prepared  in vitro  using mMessage mMachine kits(Ambion, Austin, TX). Concentrations of cDNAs and cRNAswere calculated by spectrophotometry. Oocyte Removal and Injection— Oocytes were removed sur-gically from  Xenopus laevis  as described except that a higherconcentration (0.26 mg/ml) of collagenase type IA (Sigma) wasusedtoobtainoocytesoptimalfortheautomatedvoltageclampinstrument described later.Oocyte injections were performed within 48 h after surgery.Oocytes were injected with 20 ng of    2-  4-  2-  4 concatamercRNA and free single subunit at a 1:1 ratio except with  6 and  2 subunits. To express (  4  2) 2  6 and (  4  2) 2  2 in oocyteswith high enough currents, 40 ng of total cRNA was injected ineach oocyte at a 1:1 ratio of concatamer   2-  4-  2-  4 to   6subunit, and 30 ng of total cRNA was injected at a 2:1 ratio of concatamer   2-  4-  2-  4 to   2 subunit. Dimeric concatamer  2-  4 produced larger currents than tetrameric concatamerswhen expressed with free subunits. To obtain functionalresponses above 0.1   A but below 20   A, 1.25 ng of dimericconcatamer was co-injected with 1.25 ng of free subunit (  2,  3,  4, and  4) per oocyte. 10 ng of total cRNAs were injectedfor the  6 or  2 subunit when co-expressed with  2-  4. Func-tion was assayed 3–7 days after injection.  Electrophysiology— Currentsinoocytesweremeasuredusinga manual two-electrode voltage clamp amplifier setup (oocyteclamp OC-725, Warner Instrument, Hamden, CT) or Opus-Xpress 6000A (Molecular Devices, Sunnyvale, CA) (30, 35).OpusXpress is an integrated system that provides automatedimpalement, voltage clamp, and drug delivery for up to eightoocytes in parallel (35). Electrodes were filled with 3  M  KCl andhad resistances of 0.5–10 megaohms for the voltage electrodeand 0.5–3 megaohms for the current electrode. Oocytes were voltage-clamped at a holding potential of   50 mV. Data werecollected and filtered at 50 Hz. 200   l of drugs were deliveredon top of oocytes for 4 s through the sidewall of the bath tominimize disturbance. Between drug applications, oocytesreceived a 30-s prewash and 223-s postwash with ND96 solu-tion(96m M NaCl,2m M KCl,1.8m M CaCl 2 ,1m M MgCl 2 ,5m M HEPES,pH7.6)plus0.5  M atropineperfusedthroughthebathat a rate of 3 ml/min unless otherwise noted.In concentration/response experiments, each oocyte re-ceived two initial control applications of ACh (300  M  for con-catamer expressed with free    subunits and 30   M  for con-catamerexpressedwithfree  subunits)followedbyapplicationofvariousconcentrationsofACh(fromlowtohigh).InMTSEAexperiments, each oocyte received2 m M MTSEAfor 60 sattherate of 0.9 ml/min and then was incubated with MTSEA for anadditional5minwhiletheND96washwasstoppedtoretainthe  AccessorySite-selectiveActivation 13908  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 290ãNUMBER 22ã MAY29,2015   a  t   U NI   VE R S I  DADDE  C HI  L E  on N o v e m b  e r  8  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om   reagentinthebathbeforebeingwashedwithbuffersolutionfor287 s. Oocytes were discarded after experiments involvingMTSEAbecausetheyhadbeencovalentlymodified.InNS9283experiments,10  M NS9283waspreappliedtooocytesexpress-ing   2-  4-  2-  4 and free subunits for 30 s at the rate of 1.8ml/min and incubated for an additional 80 s in a static bathbefore its co-application with ACh for 4 s at 3 ml/min atEC 20–40  concentrations (30   M  for    subunits, 1   M  for   2subunit, and 3  M  for  4 subunit). Preincubation with NS9283eliminates kinetic effects of NS9283 binding, leaving only thekinetics of ACh responses in nAChRs at equilibrium withNS9283. Control experiments were performed on the sameoocytes before NS9283 applications following the same proto-col in which 10   M  NS9283 was replaced with 0.1% (v/v)DMSO. Potentiation by NS9283 was calculated by increasedresponse to ACh with 10   M  NS9283  versus  response of AChco-applied with 0.1% DMSO. In the ACh concentration/re-sponse curve experiment with NS9283, 10  M  NS9283 was co-appliedwithdifferentconcentrationsofAChwithoutpreappli-cation to shorten the experiment duration.The peak amplitudes of experimental responses were calcu-lated relative to the maximum ACh response or the average of the first two control ACh responses to normalize the data andcompensate for variable expression levels among oocytes.MeanandS.E.werecalculatedfromnormalizedresponses.Thenumbersofoocytestestedarelisted.TheHillequationwasfittotheconcentration/responserelationshipusinganonlinearleastsquares curve fit method (Kaleidagraph, Abelbeck/Synergy,Reading,PA):  I  (  x )   I  max [  x n H /(  x n H  EC 50 n H )]where  I  (  x )isthepeak current measured at the agonist concentration  x ,  I  max  isthe maximum current peak at the saturating concentration,EC 50 istheagonistconcentrationrequiredtoachievehalfofthemaximum response, and  n H  is the Hill coefficient. Cell Culture and Transfection— All cells were maintained asdescribed previously (29). The human embryonic kidney tsA201 (HEK) cell lines stably expressing human   4  2 and  3  2 were described previously (8, 29, 36). To establish stablecell lines of    3  4,   2  2,   2  4, and   4  4, equal amounts of plasmid encoding appropriate    and    subunits were trans-fected into HEK cells using the FuGENE 6 transfection re-agent (Roche Diagnostics) at a ratio of 6   g of DNA/18  l of FuGENE 6/100-mm dish. A single colony expressing appropri-ate nAChRs was selected as described previously (36).  FLEXstation Experiments— For functional tests of nAChRsexpressed in HEK cells, we used a FLEXstation (MolecularDevices) bench top scanning fluorometer as described by Kuryatov   et al.  (36). To increase the expression level of    2  2and   3  2 nAChRs, the plates were incubated at 29 °C for 20 hbefore being tested. The membrane potential kit (MolecularDevices) was used according to the manufacturer’s protocols.Serial dilutions of NS9283 were manually added to the assay plate15minpriortorecording.AChdilutionswerepreparedinV-shaped96-wellplates(FisherScientificCo.)andaddedincellculture wells at 20   l/s during recording. Each data point wasaveragedfromthreetofourresponsesfromseparatewells.Thepotency and efficacy of drugs were calculated from the Hillequation described above. Results  NS9283 Potentiates   2* and    4* but Not    3* nAChRs— Asreported by others, NS9283 augments activation of nAChRscontainingthree  2orthree  4subunitspernAChRexpressedin oocytes or HEK cells but not   3* nAChRs (10, 12). Here weusednAChRsexpressedinHEKcellstovalidatethepharmaco-logical characteristics of the NS9283 that we synthesized (27).NS9283 increased activation by ACh (at EC 20–30  concentra-tion)of   2*and  4*celllinesby86–371%(Fig.1).An  2  4linewas not affected by NS9283. Cell lines may contain mixtures of subtypes, for example (  4  2) 2  4 and (  4  2) 2  2 in the   4  2line and (  2  4) 2  2 and (  2  4) 2  4 in the   2  4 line. Subtypescontaining sites at which NS9283 can bind ( e.g.   4/  4 in(  4  2) 2  4 or  2/  2 in (  2  4) 2  2) exhibit increased responseswith NS9283, but the (  2  4) 2  4 stoichiometry cannot bindNS9283 (12). We tested potentiation by NS9283 of (  2  4) 2  2nAChRsexpressedinoocytes(cRNAinjectionratioof   2to  4,4:1). NS9283 increased activation by 100   M  ACh of    2  4nAChR by 268% with an EC 50  of 5.02  1.81   M  and reachedmaximum potentiation at 30   M . This suggests that our unre-sponsive  2  4celllineexpressesmainlythe(  2  4) 2  4stoichi-ometry, whereas the responsive  4  2,  4  4, and  2  2 nAChRcell lines express the (  ) 2   stoichiometry and possibly the(  ) 2  stoichiometry. NS9283 did not potentiate activation by AChon  3  2or  3  4celllines(Fig.1).NS9283didnotactivateany   2*,  3*,or  4*nAChRbyitselfasreportedbyothers(datanot shown) (10, 12). FIGURE 1.  Chemical structure and potentiation effect of NS9283 on acti-vationofnAChRsubtypesexpressedinHEKcells. Various concentrationsofNS9283werepreappliedtocelllinesfor15minbeforeacuteapplicationof AChatEC 20–30 concentration( i.e.  4  2,0.4  M ;  4  4,1  M ;  3  2,4  M ;  3  4,5  M ;  2  2,0.4  M ;  2  4,0.8  M ).Afluorescentindicatorwasusedtorecordthemembranepotentialchanges.NS9283potentiates  4  2,  4  4,and  2  2nAChRs but not   2  4,   3  2, or   3  4 nAChR. Because NS9283 inhibited itsown potentiation at higher concentrations, we could not fit the concentra-tion/response curve into the Hill equation to obtain the exact potency ormaximumefficacyvaluesofNS9283forallnAChRsubtypestested.Therefore,abargraphsummaryispresentedhereforcomparison.NS9283reachesmax-imumpotentiationat3–30  M .MaximumnormalizedincreasesinresponsesbyNS9283comparedwithAChappliedaloneare370  22%for  4  2,143  10% for  4  4, and 86  20% for  2  2.  Error bars  represent the mean  S.E.  AccessorySite-selectiveActivation MAY29,2015ã VOLUME 290ãNUMBER 22  JOURNAL OF BIOLOGICAL CHEMISTRY   13909   a  t   U NI   VE R S I  DADDE  C HI  L E  on N o v e m b  e r  8  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om    NS9283 Increases Activation by Agonists— Both sazetidineandcytisinearepartialagonistsfor  4  2expressedinHEKcells(20). Our   4  2 cell line expresses a mixture of (  4  2) 2  4 and(  4  2) 2  2 stoichiometries as illustrated by the biphasic activa-tion curve by ACh alone in Fig. 2  A  (8). Partial agonism of saze-tidine arises from its exclusive and high affinity action at the  4/  2 agonist sites;  i.e.  sazetidine is a primary SSAg (21, 22).Cytisineisanintrinsicpartialagonistthatnon-selectivelybindsto both the  4/  2 and  4/  4 agonist sites (20). ACh is definedas a full agonist for both the   4/  4 site and   4/  2 site. Weinvestigated whether NS9283, which is an accessory SSAg,assists activation by ACh, sazetidine, and cytisine. At low con-centrationsofACh,NS9283fromthe  4/  4siteincreasedacti- vation of    4  2 nAChRs, but at high concentrations of ACh,NS9283didnotexceedtheefficacyofACh(18).ThisisbecauseNS9283cannotcompetewithAChtobindtothe  4/  4sites,orifitdoes,itisfunctionallyindistinguishablefromAChboundtothis site.NS9283 increases activation of nAChRs by partial agonists,but its potentiation profiles differ between the   4/  2 primary SSAg sazetidine and the non-selective partial agonist cytisine.NS9283 increased the efficacy of both sazetidine and cytisine(Fig. 2,  B  and  C  ). Interestingly, NS9283 made sazetidine a fullagonist at  4  2 nAChRs without changing its potency. This ismostlikelybecauseNS9283activatesthe  4/  4site(thecriticalaspect of potentiation), a site that is not bound by sazetidine.NS9283(10  M )alsopotentiatedactivationof   4  2nAChRsby cytisineupto10  M butnotathigherconcentrationsofcytisine(Fig. 2 C  ). Because NS9283 is an agonist at the   4/  4 site andcytisine is a partial agonist at both   4/  2 and   4/  4 sites,NS9283 likely augments activation by cytisine at   4/  2 sites,whereas increasing concentrations of cytisine compete withNS9283 for binding to the   4/  4 site, thereby preventing thefull agonist effect of NS9283 at this site. Channel block by cyti-sinecouldalsocontributetoreducedresponsesathighconcen-trations of cytisine. FIGURE2. NS9283potentiatesactivationof   4  2nAChRsbyACh,sazetidine,andcytisinedifferently. 10  M NS9283wasincubatedfor15minwithHEK cellsstablyexpressing  4  2nAChRsbeforeacuteapplicationofvariousconcentrationsofsazetidineorcytisine.Afluorescentindicatorwasusedtorecordthemembranepotentialchanges.  A ,concentration/responsecurvesofAChwithandwithoutNS9283.Notethetwo-componentcurvewithACh,reflectingahighaffinitycontributionfromthe  4/  2sitesandalowaffinitycontributionfromthe  4/  4site.TheefficacyofAChis49.9  7.1%,andtheEC 50 is0.0626  0.0099  M  for the   4/  2 sites. The efficacy for the low sensitivity   4/  4 sites is 49.1  8.5%, and the EC 50  is 3.89  1.35   M . After the   4/  4 site is occupied bypreappliedNS9283,sensitivityreflectsacutelyappliedAChactivatingathighaffinity  4/  2sites.TheefficacyofAChwithNS9283is102  1%,andtheEC 50 is0.0314  0.0024  M . B ,concentration/responsecurvesofsazetidinewithandwithoutNS9283.Theefficacyofsazetidinealoneis43.5  2.1%,andtheEC 50  is0.00262  0.00047   M . The efficacy of sazetidine with NS9283 is 94.0  2.6%, and the EC 50  is 0.00107  0.00012   M .  C  , concentration/response curves of cytisinewithandwithoutpreapplicationof10  M NS9283.Themaximumefficacyofcytisinealone(  90  M )is34.3  2.0%,andtheconcentration/responsecurvedoesnotreachplateauevenat90  M .TheefficacyofcytisinewithNS9283is57.9  1.9%,andtheEC 50 is0.0259  0.0044  M .Responsesof90  M cytisinewith NS9283 plunge to 41.3  3.0% of the maximum responses evoked by ACh.  Error bars  represent the mean  S.E.  AccessorySite-selectiveActivation 13910  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 290ãNUMBER 22ã MAY29,2015   a  t   U NI   VE R S I  DADDE  C HI  L E  on N o v e m b  e r  8  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om 
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