Synaptotagmin-1 functions as a Ca 2+ sensor for spontaneous release

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1 Synptotgmin- funtions s C + sensor for spontneous relese Jun Xu,, Zhiping P Png, Ok-Ho Shin, & Thoms C Sühof 9 Nture Ameri, In. All rights reserve. Spontneous mini relese ours t ll synpses, ut its nture remins enigmti. We foun tht > 9% of spontneous relese in murine ortil neurons ws inue y C -ining to synptotgmin- (Syt), the C sensor for fst synhronous neurotrnsmitter relese. Thus, spontneous n evoke relese use the sme C -epenent relese mehnism. As onsequene, Syt muttions tht ltere its C ffinity ltere spontneous n evoke relese orresponingly. Proxilly, Syt eletions (s oppose to point muttions) mssively inrese spontneous relese. This inrese spontneous relese remine C epenent ut ws tivte t lower C onentrtions n with lower C oopertivity thn synptotgmin-riven spontneous relese. Thus, in ition to serving s C sensor for spontneous n evoke relese, Syt lmpe seon, more sensitive C sensor for spontneous relese tht resemles the C sensor for evoke synhronous relese. These t suggest tht Syt ontrols oth evoke n spontneous relese t synpse s simultneous C -epenent tivtor n lmp of exoytosis. Spontneous miniture relese (minis) ws oserve in initil reorings of synpti tivity n hs engge neurosientists ever sine. Uner physiologil onitions, every synpse spontneously releses neurotrnsmitter qunt t low frequeny. In typil entrl neuron, these qunt up to sizle signl euse thousns of synpses re present on the neuron. A mjor question hs een whether spontneous neurotrnsmitter relese is iologilly meningful or ientl. Inee, the very nture of spontneous relese remins unler; is it truly spontneous or is it regulte,? Does it operte with the sme synpti mhinery s evoke relese, or oes it represent ifferent type of exoytosis tht involves speil synpti vesile pool,? Presynpti mini relese proues multiple postsynpti effets; for exmple, spontneous relese mintins enriti spines 7, regultes ereellr interneuron firing n suppresses lol enriti protein synthesis 9. Moreover, mny neurotrnsmitters n psyhotive ompouns, suh s enonninois, ffeine n niotine, moulte spontneous relese y tivting presynpti reeptors, initing tht spontneous minis perform iologil funtion. Consistent with this notion, sustntil omponent of spontneous relese ppers to e C + epenent n my e triggere y presynpti C + sprks 7,. An itionl, sizle omponent of spontneous minis, however, ws foun to e C + inepenent in t lest some experiments, lthough the mehnisms involve remin unler. Evoke neurotrnsmitter relese is meite y synpti memrne fusion mhinery ompose of solule NSF-tthment protein reeptors (SNAREs) n Se/Mun-like proteins, n is ontrolle y C + ining to Syt, synptotgmin- (Syt) or synptotgmin-9, whih perform similr interhngele funtions in evoke relese 9. Deletions of SNAREs n Se/Mun-like proteins lok evoke n spontneous relese, suggesting tht the generi synpti memrne fusion mhinery meites oth types of relese. Deletions of Syt n Syt, however, proue inreses in spontneous mini relese in Drosophil neuromusulr juntions n in verterte entrl synpses (lthough not in verterte utpses 9 ). This fining le to the hypothesis tht Syt is not only the C + sensor for evoke relese ut is lso lmp of SNAREs tht limits spontneous relese, n tht minis re ientl leks of this lmping funtion,.the lmping hypothesis, however, rgues ginst the notion tht spontneous relese my e iologilly meningful, s it is iffiult to imgine how n ientl yprout of evoke relese oul ontrol physiologil proess. Moreover, the lmping hypothesis fils to explin why t lest some mini relese is C + epenent. We explore how C + regultes spontneous relese n exmine the reltionship of this mehnism to the lmping of spontneous relese y synptotgmins. We foun tht C + triggere most spontneous relese, s it i most evoke relese, y ining to synptotgmin n tht eletion of synptotgmin tivte seon C + sensor for spontneous relese tht hs higher pprent C + ffinity thn synptotgmin. As result, eletion of synptotgmin le to proxil inrese in spontneous relese meite y this seon C + sensor, whih is normlly lmpe y synptotgmin. Deprtment of Moleulr & Cellulr Physiology, n Howr Hughes Meil Institute, Stnfor University, Plo Alto, Cliforni, USA. Deprtment of Neurosiene, Howr Hughes Meil Institute, n Deprtment of Moleulr Genetis, The University of Texs Southwestern Meil Center, Dlls, Texs, USA. Present ress: GlxoSmithKline (Chin) R&D Co. Puong, Shnghi, Chin (J.X.), n Deprtment of Neurosiene & Cell Biology, University of Texs Meil Brnh, Glveston, Texs, USA (O.-H.S.). Corresponene shoul e resse to T.C.S. (ts@stnfor.eu). Reeive 7 Deemer ; epte Mrh 9; pulishe online My 9; oi:./nn. NATURE NEUROSCIENCE VOLUME [ NUMBER [ JUNE 9 79

2 9 Nture Ameri, In. All rights reserve. e Surose mm C + mm C + mm C + + BAPTA-AM mm C + + thpsigrgin mm C + + thpsigrgin + BAPTA-AM pa s + Syt resue + BAPTA-AM pa BAPTA-AM Surose RRP (synpti hrge trnsfer in nc) RESULTS Spontneous relese is C + epenent Using ulture ortil neurons, we mesure evoke inhiitory postsynpti urrents (IPSCs) n spontneous miniture IPSCs (mipscs) in extrellulr meium ontining or lking C +. Removl of extrellulr C + only prtly epresse the frequeny of spontneous mipscs, ut lmost ompletely loke evoke IPSCs (Fig., n t not shown). Thus, s previously shown, spontneous mini relese is less sensitive thn evoke relese to reutions in extrellulr C + onentrtion. However, when we pre-inute ulture neurons with,-is-(o-minophenoxy)-ethne-n,n,n,n tetreti i, tetretoxymethyl ester (BAPTA-AM), memrnepermele C + heltor, lmost ll mipscs (9%) were loke (Fig., n Supplementry Fig. online). The ition of thpsigrgin to neurons in C + -free meium strongly enhne the mini frequeny, ut pre-inution with BAPTA-AM gin loke spontneous mini relese (Fig.,). Cffeine, whih ts to inrese presynpti C + onentrtions, lso inrese the frequeny of spontneous relese (Supplementry Fig. online). As esrie ove, ortil synpses lking Syt show lmost no evoke relese ut inste hve proxil inrese in spontneous relese 7, whih is thought to reflet lmping of SNARE omplexes y Syt (refs.,). This inrese spontneous relese, together with the loss of evoke relese, oul e resue y the expression of exogenous Syt n ws therefore not evelopmentlly inue (Fig.,). However, the C + requirement of mini relese in wiltype synpses rises the question whether the inrese minis in Syt- efiient synpses my lso e C + epenent. Inee, we foun tht s / BAPTA-AM Syt Surose Surose. na s f C + (mm): BAPTA-AM: + + Thpsigrgin: + + BAPTA-AM n.s. Syt resue n.s. Syt KO BAPTA-AM BAPTA-AM Figure Spontneous miniture relese is C + epenent in wil-type n Syt knokout synpses. ( ) Representtive tres (,) n summry grphs (,) of mipscs monitore y whole-ell reorings in ulture ortil neurons from wil-type () n Syt / mie uner the inite onitions ( mm BAPTA-AM pplie t 7 C for h efore n uring reorings; mm thpsigrgin e min fter stle reorings were estlishe). All reorings from ulture neurons were otine t in vitro (DIV), ll mini reorings were proue in mm TTX, n mipscs were monitore in the presene of mm D(-)--mino--phosphonovleri i (AP) n mm -yno-7-nitroquinoxline-,-ione (CNQX). Resue of Syt / neurons ws performe y lentivirl infetion t DIV. (e,f) Representtive tres (e) n summry grphs (f) of IPSCs inue y puffing. M surose onto neurons from n Syt / mie without or with pretretment with BAPTA-AM. Reoring onitions were the sme s in. Summry grphs epit the synpti hrge trnsfer integrte over s to estimte the size of the RRP. Dt shown in pnels, n f re mens ± s.e.m. (see Supplementry Tle online for ll numeril vlues). P o., n.s. ¼ nonsignifint (P.). 9% of mipscs in Syt-efiient synpses were suppresse y BAPTA-AM (Fig.,), initing tht minis re C + triggere n nnot e result of simple popping of the SNARE ssemly for fusion. A potentil onern for these experiments is tht C + my e require for the viility of neurons n the integrity of synpses. To ensure tht the unexpetely strong suppression of mipscs y BAPTA- AM speifilly reflets the effet of withrwing C + on mipscs n is not result of simple loss of ll synpti funtion, we mesure the reily relesle pool (RRP) of vesiles in synpses tht were preinute with BAPTA-AM. As estimte y pplition of hypertoni surose, we foun no erese in the size of the RRP, whih we mesure s the totl synpti hrge trnsfer tht ws inue y hypertoni surose fter pre-inution with BAPTA-AM (Fig. e,f). This result lso eliminte the possiility of efets in postsynpti GABA A reeptors fter tretment with BAPTA-AM. Spontneous relese in Syt knokout synpses These results rise severl questions. First, re minis in wil-type n Syt-efiient synpses proue y n ientil C + -epenent proess tht is simply enhne y the Syt knokout or is there funmentl ifferene in the C + triggering of minis etween wiltype n Syt-efiient synpses? Seon, o these results pply eqully to exittory n inhiitory synpses? Thir, wht is/re the C + sensor(s) for spontneous mini relese? To ress the first two questions, we mesure the C + epenene of spontneous relese in wil-type n Syt-efiient synpses in oth exittory n inhiitory synpses (Fig. ). We nlyze ulture ortil neurons from littermte wil-type n Syt / mie t inresing onentrtions of extrellulr C + n monitore miniture exittory postsynpti urrents (mepscs) n mipscs in tetrootoxin (TTX) fter phrmologil isoltion. We oserve similr ehvior of spontneous mini relese in exittory n inhiitory synpses, ut there ws mrke ifferene etween wil-type n Syt / synpses. At ll of the C + onentrtions, the mepsc n mipsc frequenies were strongly inrese in Syt / synpses, with n pprent left shift in the C + -onentrtion epenene (Fig.,). To quntify this, we fitte the results of iniviul experiments to Hill funtion n etermine the pprent C + oopertivity (Hill oeffiient) n pprent C + ffinity (K ) of extrellulr C + for miniture relese. Anlysis of multiple inepenent experiments revele signifint erese in the C + oopertivity n lrge inrese in the C + -ffinity (tht is, threefol 7 VOLUME [ NUMBER [ JUNE 9 NATURE NEUROSCIENCE

3 mipscs mm C + + mm EGTA. mm C +. mm C + Figure Enhne frequeny, inrese pprent C + ffinity n erese C + oopertivity of spontneous relese in Syt / synpses. ( ) C + epenene of the frequeny of mipscs n mepscs. Representtive mipsc tres (, see Supplementry Fig. for mepsc tres) n summry grphs (,) ofthec + epenene of the mipsc n mepsc frequeny re shown (ontinuous lines ¼ Hill funtion fits; otte lines ¼ sle C + epenene). (,e) Men pprent C + oopertivity n C + ffinity of mini relese t inhiitory () n exittory (e) synpses etermine y Hill funtion fitting to iniviul experiments. Dt shown in e re mens ± s.e.m. (see Supplementry Tle for ll numeril vlues). P o.. 9 Nture Ameri, In. All rights reserve. Mini frequeny (Hz) C + oopertivity..... [C + ] (mm) mipscs C + ffinity K (mm) pa erese in K ) of spontneous mini relese in Syt / synpses (P o., note tht the pprent C + oopertivity n C + ffinity of relese etermine y vrying the extrellulr C + -onentrtion, s performe for this stuy, unerestimtes the true oopertivity n ffinity s mesure y intrellulr C +, n is only ment s reltive mesure of these prmeters, whih nnot e trnslte iretly into intrellulr C + ffinities n oopertivities; Fig.,e). It is notle tht even though the reltive inrese in mini relese proue y Syt knokout iffere mrkely etween exittory n inhiitory synpses (mipscs, Btwofol inrese; mepscs, Bfourfol inrese), the pprent C + ffinity n C + oopertivity in wil-type n Syt / mie were lmost sle mipscs mm C + mm C + mm C + mm C + e C + oopertivity mepscs s.. [C + ] (mm)... mepscs C + ffinity K (mm) sle ientil etween the two types of synpses. These results, mong others, vlite our pproh. Our results nswer the first two of the three questions tht we pose ove; C + triggering of spontneous relese is inee funmentlly ifferent etween wil-type n Syt-efiient synpses, n this is similrly true for exittory n inhiitory synpses. In regrs to the thir question, the ientity of the C + sensor(s) involve, it is notle tht the C + -epenent properties of spontneous n evoke relese in wil-type versus Syt / synpses pper to e very similr. Speifilly, oth spontneous n evoke relese showe lower pprent C + ffinity, ut h higher pprent C + oopertivity in wil-type synpses thn in Syt / synpses. Beuse Syt serves s the primry C + sensor for evoke relese in ortil wil-type synpses, wheres n s yet unientifie C + sensor for synhronous relese ssumes this role in Syt / synpses 7,9,itistemptingto speulte tht Syt lso serves s the primry C + sensor for spontneous relese n tht the unknown synhronous C + sensor opts this role in the Syt / synpses. This hypothesis implies tht Syt n the synhronous C + sensor oth t on prime vesiles n tht the former normlly lmps the ltter (Supplementry Fig. online). This hypothesis preits tht the C + ffinity of Syt shoul itte the mgnitue of spontneous n evoke relese in wil-type synpses, preition tht n e reily teste. Knokin muttions in Syt C + -ining sites To test the hypothesis tht mipscs n mepscs re triggere y C + ining to Syt, we employe mie rrying knokin point muttions in the C + -ining site of the Syt CA omin,. Three ifferent muttions were nlyze: DN, DN n RQ (referre to s DN, DN, n RQ, respetively; Fig. ). Previous stuies hve emonstrte tht these muttions seletively lter the C + -ining properties of Syt. Speifilly, the DN muttion enhnes C + -epenent ining of Syt to SNARE omplexes without ltering its phospholipi-ining properties, wheres the DN n RQ muttions moertely (DN) or severely (RQ) erese the pprent C + ffinity of phospholipi ining y Syt without ltering SNARE omplex ining,. Moreover, the RQ muttion ws shown to lter the pprent C + ffinity of relese orresponingly, n oservtion tht provie the forml proof of the funtion of Syt s C + sensor for relese, wheres the effets of the other muttions on the pprent C + ffinity n C + oopertivity of relese were not teste. Thus, the vrious knokin mie rrying the three ifferent point muttions in Syt provie tool for testing whether Syt hs equivlent funtions s C + sensor in evoke n in spontneous relese. We first mesure the effets of the three knokin muttions on evoke relese y ompring neurons from mutnt n littermte ontrol mie in the sme experiments. We foun tht the three knokin point muttions h istint effets on the pprent C + ffinity of relese tht orrelte with their iohemil effets; the DN muttion NATURE NEUROSCIENCE VOLUME [ NUMBER [ JUNE 9 7

4 Memrne Synptotgmin- N [C+]:. mm ARTICLES mm + C+ CA C CB mm C Figure Knokin muttions ltering the pprent C+ ffinity of Syt use orresponing hnges in the pprent C+ ffinity of evoke relese. () Point muttions introue into the CA omin of mouse Syt y knokin,. The DN muttion inreses C+-epenent ining of Syt to SNARE omplexes, wheres the DN n RQ muttions erese the pprent C+ ffinity of Syt-phospholipi memrne omplexes,. () Representtive tres of evoke IPSCs monitore in ulture neurons from littermte n Syt DN, DN n RQ knokin mie. mt, mutnt. () Men IPSC mplitues in neurons from n knokin mie plotte s funtion of the extrellulr C+ onentrtion. () Men K for extrellulr C+ of IPSCs, etermine y Hill funtion fits to iniviul C+-titrtion experiments s shown in n. In ll experiments with knokin mie, eh mutnt ws nlyze with its own seprte littermte ontrol. Dt re mens ± s.e.m. (see Supplementry Tle for ll numeril vlues, for other prmeters, see Supplementry Fig. ). P o.. DN = DN DN = DN RQ = RQ mm mm DN DN mt DN RQ RQ mt na ms DN DN RQ RQ mt DN mt K for C+ (mm) Mutnt + R Q D N. D N IPSC mplitue (na) [C ] (mm) inrese the pprent C+ ffinity (tht is, erese the pprent K for C+), wheres the DN n the RQ muttions erese the pprent C+ ffinity of relese (tht is, inrese the pprent K for C+; Fig. ). None of the muttions signifintly ltere the pprent C+ oopertivity of relese, s expete (P.; Supplementry Fig. online). We then mesure the effets of the three knokin muttions on spontneous relese n oserve the sme result, initing tht miniture relese is triggere y C+ ining to Syt (Fig. n Supplementry Fig. ). The inrese in mini frequeny tht we oserve in knokin mie ontining the DN Syt muttion tht inreses its pprent C+ ffinity oul e fully loke y pre-inution with BAPTA-AM, emonstrting tht the orresponing mini relese onforms to the sme overll mehnism s in wil-type synpses (Supplementry Fig. online). Thus, spontneous relese is normlly riven y C+ ining to Syt in ulture ortil neurons. Finlly, to investigte whether the role of Syt s C+ sensor for spontneous relese opertes uring physiologilly meningful regultion of spontneous relese, we monitore the effet of niotine on spontneous relese in rin slies from wil-type n knokin mie. Niotine is known to in to, mong others, presynpti etylholine reeptors n therey regulte spontneous relese, whih we onfirme for wil-type synpses (Fig.,). In synpses from Syt knokin mutnt mie, spontneous relese ws still inrese y niotine, ut in the ontext of the overll regultion of relese y Syt; tht is, the overll mount of spontneous relese ontinue to e itte y the pprent C+ ffinity of Syt tht ws present in the synpses exmine (Fig.,). This result suggests tht the Sytepenent regultion of spontneous relese opertes uner physiologil onitions. Syt C+-ining sites lmp spontneous relese How oes eletion of Syt tivte (tht is, erepress) C+ triggering of mini relese t C+ onentrtions tht re lower thn those tht tivte Syt itself? To explore this question, we infete ulture + [C ] DN. mm DN mt DN RQ RQ mt. mm. mm mm 7 mm mm pa s Mutnt DN DN RQ DN mt RQ mt * VOLUME. [ [C+] (mm) NUMBER [ N D N R Q. D Figure Knokin muttions ltering the pprent C+ ffinity of Syt use orresponing hnges in the pprent C+ ffinity of spontneous relese. () Representtive mipsc tres monitore t ifferent C+ onentrtions in neurons ulture from littermte n knokin mie with the inite muttions. () Men mipsc frequeny plotte s funtion of the extrellulr C+ onentrtion. () Men K for extrellulr C+ for mipscs s etermine y C+ titrtions. Dt re mens ± s.e.m. (see Supplementry Tle for ll numeril vlues). P o., P o.. mm K for C+ (mm) Effet of Syt knokin muttions in slies A potentil onern of the experiments esrie ove is tht they were performe in ulture neurons in whih norml C+ trnsients oul e generte. To ress this onern, we mesure the effets of the three knokin muttions on spontneous mini relese in ute rin slies from Syt knokin mutnt mie n their littermte wiltype ontrols (Fig.,). We oserve the sme effet of the knokin muttions s in ulture neurons, initing tht Syt funtions s the C+ sensor for spontneous relese in wil-type synpses. 9 Nture Ameri, In. All rights reserve. 7 JUNE 9 NATURE NEUROSCIENCE

5 9 Nture Ameri, In. All rights reserve. mipsc frequeny (Hz) DN DN mt DN RQ RQ mt Niotine Niotine DN DN mt pa DN pa Niotine: DN DN RQ s neurons from Syt / mie with ontrol lentivirus or lentiviruses expressing wil-type or mutnt Syt in whih the C + -ining sites of either the CA omin, the CB omin or oth of the C omins of Syt were mutte (Fig. ). In these mutnts, three sprtte resiues in the respetive C + -ining sites of the CA n/or the CB omin were exhnge for lnine resiues, therey olishing C + ining to the C omins. The mutnt C omins were expresse t similr levels in the infete neurons n still fole well (Supplementry Fig. online n t not shown), llowing us to etermine the effet of C + ining to the iniviul C omins on the C + triggering n lmping of spontneous exoytosis. As expete 7, evoke IPSCs were impire in Syt / neurons ut were fully resue y expression of wil-type Syt (Fig. ). Muttion of the C + -ining sites of the CA omin prtilly loke resue of evoke responses (% erese), wheres muttion of the CB omin or of oth C omins ompletely loke resue of evoke relese (Fig. ). This result is onsistent with previous stuies tht foun tht C + ining to oth the CA n the CB omin is involve in the C + triggering of synhronous relese, with the CB omin eing reltively more importnt,. Notly, the muttions of the C omin C + -ining sites lso impire the ility of Syt to lmp spontneous relese (Fig. n Supplementry Fig. 7 online). However, the reltive effiy of ifferent mutnts iffere etween Mutnt DN * DN RQ RQ mt Mutnt RQ s * Figure Knokin muttions in Syt lter spontneous relese monitore in ute slies. (,) Representtive tres (, lirtion rs t the ottom pply to ll tres ove the rs) n summry grphs () of mipscs reore in ute ortil slies from littermte n Syt knokin mie. Note tht the inrese frequeny of spontneous relese in DN mutnt mie ws fully loke y BAPTA-AM (Supplementry Fig. ). (,) Representtive tres () n frequeny summry grphs () of mipscs monitore in ute ortil slies n Syt knokin mie efore n fter ition of mm niotine, pplie t perfusion rte of ml min t min fter stle reorings were estlishe. Dt re mens ± s.e.m. (see Supplementry Tle for ll numeril vlues n sttistil nlyses of ). * P o., P o., P o.. evoke n spontneous relese in tht the CA omin muttion ws more eleterious thn the CB omin muttion for the lmping funtion of Syt (spontneous mini frequeny: CB omin mutnt, B Hz;CA mutnt,b Hz), wheres the opposite ws oserve for the relese funtion of Syt (Fig. n Supplementry Fig. 7). To more preisely quntify the similrity of the effet of the CA n CB muttions on miniture relese, we mesure the C + epenene of the mipsc frequeny in Syt knokout neurons expressing wil-type Syt or the CA or CB omin mutnt of Syt (Fig. f n Supplementry Fig. online). Quntittion of the pprent C + ffinity n C + oopertivity of spontneous relese revele tht neither the CA nor the CB muttion resue the hnge in either prmeter (Fig. e,f), espite similr expression levels. These results suggest tht, in ition to triggering synhronous n spontneous relese, the C + -ining sites of oth C omins re involve in lmping the synhronous C + sensor, with the CA omin eing more effetive thn the CB omin t lmping the seon C + sensor n the CB omin eing more effetive thn the CA omin t triggering relese. C + triggering n lmping of relese A plusile hypothesis to ount for the requirement of the C omin C + -ining sites for lmping mini relese is tht these sites lmp mini relese in C + -inepenent mnner ut tivte evoke n spontneous relese in C + -epenent mnner. It is likely tht the ility of Syt to evoke synhronous relese requires lose proximity of the Syt C omins to the memrne in whih the SNAREs re lote euse, s onfirme y our experiments on the knokin muttions (Figs. ), Syt funtions y simultneous, rpi n C + -epenent ining to SNARE omplexes n to phospholipis. Inee, most notle feture of synptotgmin-triggere relese is the spee with whih it opertes uring n tion potentil, whih n est e ounte for y lk of onformtionl hnges involve n y short retion istnes. This rgument shoul not pply to the funtion of Syt s lmp of the synhronous C + sensor or s C + sensor for spontneous relese, s these funtions o not involve trnsient C + fluxes tht re inue y tion potentils. To test these preitions, we exmine the effet of inresing the istne of the Syt C omins from the vesile memrne on spontneous n evoke relese (Fig. 7). We expresse Syt with uplite linker sequene etween the C omins n trnsmemrne region. The oule-linker mutnt resue only % of evoke C + triggere relese, ut i not lter the pprent C + ffinity or C + oopertivity of evoke relese s expete, s the C omins were not hnge (Fig. 7,, n Supplementry Fig. 9 online). Moreover, the oule-linker muttion i not lter the lmping funtion of Syt (Fig. 7,e), s we preite. Thus, lthough oth evoke n spontneous relese normlly utilize Syt s C + sensor, only the former requires rpi oupling of the C omins to the NATURE NEUROSCIENCE VOLUME [ NUMBER [ JUNE 9 7

6 9 Nture Ameri, In. All rights reserve. Evoke IPSC mpl. (na) e C + oopertivity N Memrne resue: memrne euse it is epenent on the C + ynmis inue y n tion potentil, wheres the ltter opertes vi rnom sensing of C + sprks n lmping of the synhronous C + sensor. DISCUSSION Evoke n spontneous neurotrnsmitter relese re generlly onsiere to represent istint types of relese tht re ifferentilly regulte. Their istint ntures re eviene y the ft tht resue: Ctrl Syt Syt CA DA f C + onentrtion (mm) Ctrl Syt CA DA DA C + C + CA Syt CB DA Syt CAB DA Syt CB DA CB C K for C + (mm) CA DA = D7A, DA, DA CB DA = D9A, DA, DA CAB DA = CA DA + CB DA.... resue: resue: Ctrl Syt + CA DA + CB DA Syt CA DA Syt CB DA Ctrl Syt CA DA Syt CB DA Syt CAB DA Figure Syt lmping of spontneous relese requires intt Syt C + -ining sites. () Digrm of muttions in the CA n/or the CB omin of Syt tht olish C + ining to iniviul C omins. Eh muttion onsists of three seprte sprtte-to-lnine sustitutions in the C + -ining site (referre to s DA muttions), with the preise sustitutions liste on the right. Muttions were esigne on the sis of the tomi strutures of the CA n CB omins,.(,) Comprison of the evoke IPSC mplitue () n spontneous mipsc frequeny () in neurons from littermte or Syt / mie. Syt / neurons were infete with either ontrol lentivirus (Ctrl), lentivirus expressing Syt (Syt) or lentivirus expressing mutnt Syt (Syt CA DA, mutnt lking the CA omin C + -ining sites; Syt CB DA, mutnt lking the CB omin C + -ining sites; Syt CAB DA, mutnt lking the CA n the CB omin C + -ining sites; see Supplementry Figs. for immunolots n representtive tres). () Men spontneous mipsc frequeny s funtion of the extrellulr C + onentrtion in or Syt / neurons expressing CA DA or CB DA mutnt Syt (ontinuous lines, fittings with Hill funtions; otte lines, sle fittings; see Supplementry Fig. for representtive tres). (e,f) Summry grphs of the men C + oopertivity (e) nk for extrellulr C + (f) etermine from the t shown in. Dt shown re mens ± s.e.m. (see Supplementry Tle for ll numeril vlues). P o., n.s. ¼ nonsignifint (P.). spontneous relese is mintine in the presene of the soiumhnnel inhiitor TTX, whih olishes tion potentils n evoke relese. We foun, however, tht espite their ifferentil regultion, these two types of relese re mehnistilly ientil in tht they oth re triggere y C + ining to Syt (Figs. ). The mjor eviene for this onlusion rests on the three Syt knokin muttions tht we use. We previously emonstrte tht these muttions either inrese C + -epenent ining of Syt to SNARE omplexes (DN) or erese the pprent C + ffinity of Syt ining to phospholipis (DN n RQ),. In iret omprison of ll three knokin muttions, we foun tht they use orresponing hnge in evoke relese n preisely equivlent hnge in spontneous relese (Figs. ). The fining tht Syt funtions s C + sensor for oth spontneous n evoke relese ws surprising to us euse Syt is thought to e physiologilly tivte only y C + influx uring n tion potentil, s the C + ffinity of Syt is too low for it to e tivte y sumiromolr resting C + onentrtions. Inee, it is likely tht spontneous relese is triggere not y the resting C + onentrtion in the presynpti terminl, ut y C + sprks 7,.C + sprks my rise from internl stores or C + influx, s inite y the inrese in spontneous relese inue y thpsigrgin, niotine n ffeine n the epenene of spontneous relese on extr- n intrellulr C + (Figs., n n Supplementry Fig. ). The Syt knokin muttions use similr hnges in spontneous relese in neurons in ulture (Figs. n ) n in ute rin slies (Fig. ), suggesting tht the role of Syt s C + sensor for spontneous relese is not peulir to ulture neurons, ut is generlly pplile. Moreover, our results support previous suggestions 7 9 tht spontneous relese is physiologilly importnt. C + regultion generlly implies physiologilly ontrolle funtion; thus, the fining tht spontneous relese is ontrolle y C + ining to Syt implies physiologil role, s is lso supporte y the oservtion tht the potentition of spontneous relese y niotine operte vi C + ining to Syt (Fig. ). Mny neurotrnsmitters n neuromoultors t y inresing or eresing presynptil C + onentrtions, suggesting tht these gents my ontrol synpti iruits, t lest in prt, y regulting Syt- epenent spontneous relese without triggering tion potentils. A mjor rgument ginst the notion tht Syt n other synptotgmins re C + sensors for spontneous relese ws the fining tht 7 VOLUME [ NUMBER [ JUNE 9 NATURE NEUROSCIENCE

7 9 Nture Ameri, In. All rights reserve. N Syt linker. mm mm mm Syt linker eipsc mplitue (na) N na Memrne s C + C + CA Syt linker IPSCs mipscs pa eletions of Syt n of Syt proxilly inrese spontneous relese. We foun tht the proxil inrese in spontneous relese inue y eletion of Syt ws result of the unlmping, not of SNARE omplexes, ut of n lterntive seon C + sensor. This fining rgues ginst the notion tht Syt ts s lmp of SNARE omplexes,, notion tht ws plusile given the ining of Syt to SNARE omplexes n the ft tht SNARE omplexes re proly ssemle uring priming, rey to go for relese 9, ut is ontrite y our t. Clerly, SNARE omplexes re tight n o not simply pop euse otherwise spontneous fusion woul not e C + epenent. The seon C + sensor for spontneous relese tht is unlmpe y the Syt / hs sustntilly higher pprent C + ffinity n sustntilly lower pprent C + oopertivity thn Syt. These properties orrespon to those of the seon C + sensor for relese tht rives synhronous exoytosis n tht we previously hrterize iophysilly in the lyx of Hel synpse 7. In the lyx of Hel synpse, Syt funtions s C + sensor for synhronous evoke relese inste of Syt, n synhronous relese persists in this synpse fter knokout of Syt, similr to synhronous relese in ortil synpses fter knokout of Syt (refs.,,7). The lyx of Hel synpse llows preise mnipultions n mesurements of presynpti C + onentrtions n of exoytosis,9, whih iffers from other synpses, permitting us to efine the iophysil properties of Syt s the C + sensor for synhronous relese n of the seon C + sensor tht meites synhronous relese, lthough the ientity of this C + sensor remins unknown 7.TheseonC + sensor shows lower C + oopertivity thn synptotgmins s C + sensor for synhronous relese ut, s result of this lower C + oopertivity, shows higher pprent C + ffinity 7. These finings le to the ul C + sensor of neurotrnsmitter relese moel, oring to whih evoke CB C C + C + CA s mm CB Syt linker [C + ] (mm) e C mm [C + ] Syt L Figure 7 Memrne proximity of Syt C omins ontriutes to evoke, ut not spontneous, relese. () Digrm of insertion muttion in Syt tht uplites the linker seprting C omins from the memrne. () Representtive tres of evoke IPSCs in neurons n Syt / neurons expressing mutnt Syt with oule linker. Responses were monitore t ifferent extrellulr C + onentrtions. () Plotofthemen IPSC mplitue s funtion of the extrellulr C + onentrtion (see Supplementry Fig. 9 for summry grphs). (,e) Representtive tres () n summry grph of the frequeny (e) of mipscs monitore in neurons n in Syt / neurons expressing the oule-linker mutnt of Syt. Dt shown in ll pnels re mens ± s.e.m. (see Supplementry Tle for ll numeril vlues). P o., n.s. ¼ nonsignifint. relese is normlly meite y synptotgmins, wheres the seon C + sensor generlly mkes only minor ontriution to relese, ut is tivte uring high-frequeny stimultion 7,9,7. The most prsimonious explntion of our results is tht the sme seon C + sensor is lso responsile for the inrese spontneous relese in Syt n Syt knokout synpses. At present, we nnot rule out the possiility tht more thn two types of C + sensors meite relese (tht is, tht evoke synhronous relese uner physiologil onitions n the inrese spontneous relese fter Syt or Syt eletions re riven y istint C + sensors), ut the similrity etween the ifferent forms of relese rgues ginst this possiility. Thus, it ppers prole tht the ul C + sensor moel is generlly pplile to spontneous n evoke relese n tht ll relese is uner the ontrol of only two ompeting C + sensors: synptotgmins tht ominte uner physiologil onitions n n unknown seon C + -sensor tht kiks in when synptotgmin is elete or when synpses re stimulte repetely (Supplementry Fig. ). Although it remins unler s to how Syt n Syt lmp the seon C + sensor, our results emonstrte tht the C + -ining sites of Syt themselves re essentil for lmping. Moreover, we foun tht the CB omin of Syt ws more importnt thn the CA omin for evoke exoytosis (Fig., s reporte previously, ), ut tht the CA omin ws more importnt for lmping spontneous relese thn the CB-omin (Fig.,). Moreover, lmping i not epen on the istne of the C omins from the memrne, wheres evoke relese i (Fig. 7). Thus, two lines of eviene support the notion tht the two funtions of Syt, C + sensing versus C + lmping, re meite y relte, ut istint, mehnisms. Our t lso rise new questions. Most notly, wht is the ientity of the seon C + sensor n how oes it intert with synptotgmins? How o the C + -ining sites of Syt lmp the seon C + sensor? Beuse the seon C + sensor opertes with n pprently higher C + ffinity, this effet is unlikely to involve C + ining to Syt. Clmping must either e meite y the top C omin sequenes inepenently of C + or y C omins with prtilly oupie C + -ining sites. In the lyx synpse, we hve oserve ompetition etween the synhronous n synhronous C + sensors 7, s i previous stuies in hippompl synpses,. In ontrst, our urrent results n our previous stuies on ortil synpses 7 provie eviene for lmping of synhronous relese y the synhronous C + sensor. This issue my e relte to the puzzling sene of inrese mini relese in Syt-efiient utpses 9, ; resolving it will require the ientifition of the synhronous C + sensor. Another question is whether there is physiologil regultion of Syt tht intivtes it (n evokes relese), therey tivting spontneous mini relese, possiility tht woul onfer new ynmi imension onto synpses n ount for the physiologil role of the mehnisms tht we esrie here. Answering this question gin requires the ientifition of the synhronous C + sensor. NATURE NEUROSCIENCE VOLUME [ NUMBER [ JUNE 9 7

8 9 Nture Ameri, In. All rights reserve. METHODS Methos n ny ssoite referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk I. Kornlum, J. Mithell, L. Fn n A. Roth for exellent tehnil ssistne, n A. Mximov n C. Zhng for vie. AUTHOR CONTRIBUTIONS J.X., Z.P.P. n O.-H.S. plnne, performe n nlyze the experiments. T.C.S. oneive the projet, supervise the experiments n wrote the pper. Pulishe online t Reprints n permissions informtion is ville online t reprintsnpermissions/. Ftt, P. & Ktz, B. Spontneous suthreshol tivity t motor nerve enings. J. Physiol. (Lon.) 7, 9 (9).. Glitsh, M.D. Spontneous neurotrnsmitter relese n C + how spontneous is spontneous neurotrnsmitter relese? Cell Clium, 9 ().. Chung, C. & Kvlli, E.T. Seeking funtion for spontneous neurotrnsmission. Nt. Neurosi. 9, ().. Deither, D.L. et l. 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A gin-of-funtion muttion in synptotgmin- revels ritil role of C + -epenent SNARE-omplex ining in synpti exoytosis. J. Neurosi., ().. Mkler, J.M., Drummon, J.A., Loewen, C.A., Roinson, I.M. & Reist, N.E. The C B C + -ining motif of synptotgmin is require for synpti trnsmission in vivo. Nture, ().. Nishiki, T. & Augustine, G.J. Dul roles of the CB omin of synptotgmin I in synhronizing C + -epenent neurotrnsmitter relese. J. Neurosi., ().. Stevens, C.F. & Sullivn, J.M. The synptotgmin CA omin is prt of the lium sensor ontrolling fst synpti trnsmission. Neuron 9, 99 (). 7. Sun, J. et l. AulC + sensor moel for neurotrnsmitter relese in entrl synpse. Nture, 7 (7).. Shneggenurger, R. & Neher, E. Intrellulr lium epenene of trnsmitter relese rtes t fst entrl synpse. Nture, 9 9 (). 9. Bollmnn, J.H., Skmnn, B. & Borst, J.G. Clium sensitivity of glutmte relese in lyx-type terminl. Siene 9, 9 97 ().. Otsu, Y. et l. Competition etween phsi n synhronous relese for reovere synpti vesiles t eveloping hippompl utpti synpses. J. Neurosi., ().. Hgler, D.J. Jr. & Go, Y. Properties of synhronous n synhronous relese uring pulse trin epression in ulture hippompl neurons. J. Neurophysiol., ().. Uh, J., Zhng, X., Sho, X., Sühof, T.C. & Rizo, J. C + ining to synptotgmin: how mny C + ions in to the tip of C-omin? EMBO J. 7, 9 9 (99).. Fernnez, I. et l. Three-imensionl struture of the synptotgmin CB-omin: synptotgmin s phospholipi ining mhine. Neuron, 7 9 (). 7 VOLUME [ NUMBER [ JUNE 9 NATURE NEUROSCIENCE

9 9 Nture Ameri, In. All rights reserve. ONLINE METHODS Regents, mie n neuronl ultures. We otine AP, CNQX, iuulline n CGP from Toris, niotine n ffeine from Sigm, n EGTA-AM n BAPTA-AM from ClBioChem. Neurons were ulture s esrie previously,7 in moifie egle meium (Invitrogen) supplemente with B7 (Invitrogen), gluose, trnsferrin, fetl ovine serum n ytosine -D-rinofurnosie (Sigm). Syt knokout n knokin mie were esrie previously 9,,. All experiments were performe on either ortil neurons ulture from littermte wil-type n mutnt offspring from heterozygous rossings (for Syt knokin mie) or neurons from the sme ulture infete with the vrious test n ontrol viruses (for Syt knokout mie resue experiments). Genertion n use of reominnt lentivirus. The lentivirl onstruts tht we use were se on the pfuw vetor (moifie from pfugw vetor ), shuttle vetor ontining the gene of interest n reomintion rms for inorporting into the mmmlin genome. The vetor inlues the HIV- flp sequene, the humn polyuiquitin promoter-c, multiloning site n WRE element. Reominnt lentiviruses were proue y trnsfeting HEK 9T ells with pfuw, pvsvg n pcmvd.9 using FuGENE. Viruses were hrveste h fter trnsfetion y olleting the meium from trnsfete ells n.-mm filter ws use to remove ellulr eris. Protein expression ws teste in the trnsfete 9T ells to ensure tht viruses were working efore they were pplie to neurons. Neurons were infete with ml of onitione ell meium for eh -well of high-ensity neurons t DIV, the meium ws exhnge for norml growth meium t DIV, n ells were nlyze t DIV. Syt point muttions were generte using stnr proeures. The Syt linker onstruts were me y PCR using two sets of primers (JX: -CGG AAT TCA TGG TGA GTG CCA GTC ATC CT- n JX: -GGC AGG CAC TGC AGA AGG ACG- ; JX: -GAA AGT ATA GGA ACT TCG TCG ATC GAC CTC G- n OH: -GCT CTA GAT TAC TTC TTG ACA GCC AGC ATG GC- ). Eletrophysiologil reorings from ulture ortil neurons. We rrie out reorings essentilly s esrie,,. mipscs n eipscs were reore inthepreseneofmm CNQXnmM AP. mepscs were reore in the presene of mm iuulline n mm CGP. EPSCs were reore with pipette solution mm potssium gluonte, mm KCl, mm MgCl, mm HEPES, mm EGTA,. mm CCl,mMK ATP,. mm N GTP n mm gluose, ph juste to 7. with KOH. mipscs n mepscs were nlyze y Mini Anlysis Softwre (Synptosoft; mipsc serh prmeters: gin, ; loks,,9; threshol, pa; perio to serh for lol mximum,, ms; time efore pek for seline,, ms; perio to serh ey time,,; frtion of pek to fin ey time,.; perio to verge seline,, ms; re threshol, ; numer of points to verge for pek, ; iretion of pek, negtive; mepsc serh prmeters: gin, ; loks,,9; threshol, pa; perio to serh for lol mximum,, ms; time efore pek for seline,, ms; perio to serh ey time,,; frtion of pek to fin ey time,.; perio to verge seline,, ms; re threshol, ; numer of points to verge for pek, ; iretion of pek, negtive). The initil nlysis ws one utomtilly y the softwre with susequent visul proofreing. Applition of. M surose (in extrellulr solution) ws effete with piospritzer for s (pressure ¼ psi), with puffing pipette tip ple mmfromthesom.forc + -titrtion experiments, IPSCs, mipscs n mepsc were reore in th solutions ontining the vrious C + onentrtions ( + mm EGTA,.,.,,, n for mipsc or mepsc;.,,, n mm for IPSC), n the frequeny of mipscs or mepscs or the mplitue of the evoke IPSCs ws plotte s funtion of the C + onentrtion on logrithm xis. The urves were fitte with Hill funtion (Y ¼ VmxXn K n +X,whereV n mx is the mximl response, K is the C + ffinity n n is the oopertivity ), using IGOR Pro. softwre (Wvemetris) for the fitting, n the mximl responses, the pprent K for C + n the Hill oeffiient (tht is, pprent C + oopertivity) were erive from the fits. Eletrophysiologil reorings from ute ortil slies. Cortil slies ( mm) were prepre from mle littermte mie ge. Anesthetize mie were epitte, the rins were remove n ple into ie ol issetion uffer (7 mm NCl, mm KCl,. mm NH PO, 7 mm MgSO, mm NHCO,mMD-gluose, 7 mm surose,. mm sori i n. mm CCl ), n slies were ut with Lei virtome. The slies were inute t C in rtifiil ererospinl flui ( mm NCl, mm KCl,. mm NH PO, mm MgSO, mm NHCO,mM D-gluose n mm CCl )gssewith9%o /%CO for h, n then kept t C uner the sme onitions until they were trnsferre to the reoring hmer, whih ws perfuse t ml min with rogente rtifiil ererospinl flui ontining mm CNQX, mm APnmM TTX to mesure mipsc. Slies were equilirte for min efore reorings. All reorings were performe in lyer / pyrmil neurons of the somtosensory ortex; pyrmil neurons were ientifie y their size n single pil enrite. Reorings were otine in voltge-lmp whole-ell moe using Multilmp 7B mplifier n holing potentil of 7 mv. The whole-ell pipette solution ontine mm KCl, mm NCl, mm HEPES, mm EGTA,. mm N GTP, mm MgATP n mm QX-. The pipettes tht we use for whole-ell reoring h resistne of MO; neurons with series resistne of MO or lek urrent of pa were isre. Spontneous miniture postsynpti urrents were monitore over -min perio n the series resistne ws monitore efore n fter eh reoring. Dt were nlyze offline using Mini-Anlysis. At lest three littermte pirs of wil-type n DN, DN n RQ knokin mie were nlyze for eh prmeter. Sttistil nlyses. All sttistil omprisons were me using Stuent s t test or two-wy ANOVA s esrie in Supplementry Tle.. Lois, C., Hong, E.J., Pese, S., Brown, E.J. & Bltimore, D. Germline trnsmission n tissue-speifi expression of trnsgenes elivere y lentivirl vetors. Siene 9, 7 ().. Hn, W. et l. N-glyosyltion is essentil for vesiulr trgeting of synptotgmin. Neuron, 99 ()..Mximov, A., Png, Z.P., Tervo, D.G. & Sühof, T.C. Monitoring synpti trnsmission in primry neuronl ultures using lol extrellulr stimultion. J. Neurosi. Methos, 7 7 (7). oi:./nn. NATURE NEUROSCIENCE