Measuring [Ca2+] in the endoplasmic reticulum with aequorin

Cell Calcium (2002) 32(5–6), 251–260 0143-4160/02/$ – see on ma e © 2002 Else ie Science L d. All igh s ese ed. doi:10.1016/S0143-4160(02)00186-0 Measu ing [Ca2+] in he endoplasmic e iculum wi h aequo in J. Al a ez, M. Mon e o Depa amen o de Bioqu´ ımica y Biolog´ ıa Molecula y Fisiolog´ ıa, Facul ad de Medicina, Ins i u o de Biolog´ ıa y Gené ica Molecula (IBGM), Uni e sidad de Valladolid and Consejo Supe io de In es igaciones Cien ´ ı icas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, Spain Summa y The pho op o ein aequo in was he i s p obe used o measu e speci ically he [Ca2+] inside he lumen o he endoplasmic e iculum ([Ca2+]ER) o in ac cells and i p o ides alues o he s eady-s a e [Ca2+]ER, a ound 500␮M, ha closely ma ch hose ob ained now by o he p ocedu es. Aequo in-based me hods o measu e [Ca2+]ER o e se e al ad an ages: (i) a ge ing o he p obe is ex emely p ecise; (ii) he use o low Ca2+-a ini y aequo in allows co e ing a la ge dynamic ange o [Ca2+], om 10−5 o 10−3M; (iii) aequo in is nea ly insensi i e o changes in Mg2+o pH, has a high signal- o-noise a io and calib a ion o he esul s in [Ca2+] is made s aigh o wa d using a simple algo i hm; and (i ) he equipmen equi ed o luminescence measu emen s in cell popula ions is simple and low-cos . On he nega i e side, his echnique has also some disad an ages: (i) he ela i ely low amoun o emi ed ligh makes di icul pe o ming single-cell imaging s udies; (ii) econs i u ion o aequo in wi h coelen e azine equi es p e ious comple e deple ion o Ca2+o he ER o 1–2h, a maneu e ha may esul in dele e ious e ec s in some cells; (iii) because o he high a e o aequo in consump ion a s eady-s a e [Ca2+]ER, only ela i ely b ie expe imen s can be pe o med; and (i ) exp ession o ER- a ge ed aequo in equi es p e ious ans ec ion o in ec ion o in oduce he app op ia e DNA cons uc , o al e na i ely he use o s able cell clones. Choosing aequo in o o he echniques o measu e [Ca2+]ER will depend o he co ec balance be ween hese p ope ies in a pa icula p oblem. © 2002 Else ie Science L d. All igh s ese ed. HISTORICAL BACKGROUND Al hough he ER has been known o be he main in acel- lula Ca2+s o e o nea ly 20 yea s, moni o ing he ee [Ca2+] in he ER ([Ca2+]ER) has p o en o be a di ficul ask. The fi s s udies using low-a fini y fluo escen indica o s equi ed cell pe meabiliza ion o elease he cy osolic dye and could no a oid compa men aliza ion o he dye in o o he o ganelles [1–3]. Apa o ha , selec i i y o hese p obes o e Mg2+is poo and calib a ion was highly un- ce ain. On he o he side, use o aequo in o measu e [Ca2+]ER has equi ed sol ing wo di ficul echnical p ob- lems: ge ing specific a ge ing o he p obe in o he ER and modi ying he Ca2+-a fini y o he p obe o each he adequa e [Ca2+] ange. Recei ed 1 Sep embe 2002 Accep ed 1 Oc obe 2002 Co espondence o: D Ja ie Al a ez, Depa amen o de Bioqu´ ımica y Biol. Mol. y Fisiolog´ ıa, Facul ad de Medicina, Ramón y Cajal, 7, E-47005 Valladolid, Spain. Tel.: +34-983-423085; ax: +34-983-423588; e-mail: jal [email p o ec ed]a.es The p oblem o a ge ing Ta ge ing a p o ein o s ay in he ER equi es no only adding a N- e minal p esequence able o signal ansloca- ion in o he ER, bu also a second signal designed o make he p o ein s ay in he ER, a oiding p og ession h ough he sec e o y pa hway. The fi s app oach o a ge ing ae- quo in o he ER used he known Lys-Asp-Glu-Leu (KDEL) mo i appended o he C- e minal o aequo in o e ain he p o ein in he ER [4,5]. Howe e , his modifica ion caused a spon aneous and Ca2+independen deg ada ion o he p o ein [6,7]. In addi ion, he luminescence signal p o ided qui e low [Ca2+]ER alues (1–5 ␮M) [4], ha dly consis en wi h he ole o he ER as a Ca2+-s o e. These findings can be now explained on he basis o he high Ca2+-a fini y o he na i e aequo in used and he p esence o a small low-Ca2+compa men in he ER (see below). In o de o a oid hese p oblems, a di e en p ocedu e was designed o e ain he chime ic pho op o ein in he ER. Immunoglobulin hea y chains a e ansloca ed in o he ER and hen bind o he chape one BiP h ough i s CH1 egion un il he ligh chain a i es. Thus, in cells 251 252 J Al a ez, M Mon e o Fig. 1 Chime ic aequo in cons uc a ge ed o he endoplasmic e iculum and cloned in o he he pes simplex i us ype 1 plasmid (pHSVe AEQ). The as e isk deno es he mu a ion o one o he Ca2+-binding si es. The HA1 segmen co esponds o he hemagglu inin epi ope ag. ha do no p oduce ligh chains, a chime ic polypep ide con aining he CH1 egion is expec ed o be e ained in o he ER [8]. Tha was he case. A chime ic aequo in con aining a po ion o he immunoglobulin hea y chain gene including leade , VDJ and CH1 domains, used o he N- e minal o aequo in (Fig. 1), was co ec ly a ge ed and e ained in o he ER [9]. Elec on mic oscopy using aequo in- agged immunogold labeling confi med ha ER- a ge ing was specific and ha he pho op o ein was absen in o he s uc u es o he sec e o y pa hway such as he Golgi complex [10]. The p oblem o he Ca2+-a fini y The immunoglobulin-aequo in chime a sol ed he p ob- lem o a ge ing, bu ha was no enough o measu e [Ca2+]ER because o he high Ca2+-a fini y o na i e ae- quo in, which makes i unable o measu e [Ca2+] alues abo e 5 ␮M. Fig. 2 shows calib a ion cu es o a se ies o aequo ins. L/Lmax alues co espond o he a io among he luminescence emi ed pe second a e e y [Ca2+] and he o al luminescence han emains o be emi ed by he sample. The e o e, a alue o 0 o log L/Lmax co e- sponds o a si ua ion in which all he aequo in molecules emi hei ligh in one second, and a alue o −1 means Fig. 2 Calib a ion cu es o na i e aequo in econs i u ed wi h wild- ype coelen e azine (AEQ1) a 37◦C, mu a ed aequo in econs i u ed wi h wild- ype coelen e azine (AEQ2) a 37◦C and mu a ed aequo in econs i u ed wi h coelen e azine n (AEQ3) a bo h 22 and 37◦C. See [13,14] o mo e de ails. ha 10% o he aequo in molecules a e emi ing hei ligh e e y second. Taking in o accoun ha emission o ligh by aequo in is i e e sible (e e y aequo in molecule emi s ligh only once du ing he expe imen ), s able mea- su emen s o [Ca2+] when log L/Lmax is ≥−1 a e no pos- sible. This means ha na i e aequo in (AEQ1 in Fig. 2) can only measu e [Ca2+] alues up o abou 5 ␮M. To ex end he measu ing ange, a poin mu a ion was in o- duced (Asp119 →Ala) ha was known o educe abou 10- old he Ca2+a fini y o aequo in [11]. This e ec i ely inc eased he maximum [Ca2+] alues ha could be mea- su ed up o a ound 100 ␮M (AEQ2, Fig. 2), bu ha was s ill no enough o measu e s eady-s a e [Ca2+]ER alues. On he fi s place, emission o luminescence equi es p e- ious econs i u ion wi h he co ac o coelen e azine, bu ha dly any econs i u ion o na i e o mu a ed aequo in could be ob ained in in ac cells wi h he ER ull o Ca2+. Thus, o a oid consump ion o aequo in du ing econs i- u ion, he ER was deple ed o Ca2+be o e econs i u ion. Then, when Ca2+was ein oduced in he ex acellu- la medium, la ge peaks o luminescence we e ob ained (no ma e i na i e o mu a ed aequo in was used) ha consumed mo e han 90% o he aequo in in he fi s minu e [9,10]. Calib a ed in o [Ca2+], hese peaks co e- sponded o a [Ca2+] o abou 5 ␮M using na i e aequo in and a ound 100 ␮M using mu a ed aequo in. The heigh o hese peaks ma ched p ecisely he Ca2+-sa u a ion le el o each ype o aequo in and hese da a clea ly sug- ges ed ha he eal s eady-s a e [Ca2+]ER was highe han 100 ␮M. An ini ial al e na i e o ob ain da a on he dynamics o [Ca2+] in he ER using hese p obes was o use S 2+ asaCa 2+su oga e. S 2+beha es simila ly o Ca2+wi h espec o Ca2+pumps and mos Ca2+channels, bu i s a fini y o aequo in is 100- old smalle . This app oach allowed measu ing s eady-s a e [S 2+] alues o 1–2 mM and dynamic mo emen s o his ca ion unde di e en Cell Calcium (2002) 32(5–6), 251–260 © 2002 Else ie Science L d. All igh s ese ed. ER [Ca2+] and aequo in 253 condi ions, e.g. a as S 2+ elease a e cell s imula ion wi h agonis s p oducing inosi ol 1,4,5- isphospha e o a slow one a e ea men wi h ER-Ca2+-ATPase blocke s [9,10]. Bu he eal s eady-s a e [Ca2+]ER was s ill unclea . To measu e i , i was necessa y o educe u he he a fin- i y o aequo in o Ca2+. Tha was achie ed indi ec ly by using a semisyn he ic coelen e azine, named coelen e - azine n [12], ha educes he a e o emission o lumi- nescence by aequo in. The calib a ion cu e o mu a ed aequo in econs i u ed wi h coelen e azine n (AEQ3, Fig. 2) shows ha he ange o [Ca2+] ha can be mea- su ed wi h his p obe go om 10–20 ␮M o abou 1 mM. In addi ion, wo king a oom empe a u e (22 ◦C) educes s ill u he he a e o luminescence, allowing o mea- su e millimola le els o Ca2+ o a longe pe iod o ime. Using his app oach, we could finally ob ain measu e- men s o [Ca2+]ER dynamics in in ac HeLa cells [13,14]. A e wa ds, his new p obe has been also applied o he measu e o [Ca2+] in he sa coplasmic e iculum o skele- al muscle myo ubes, by exp essing a closely ela ed ae- quo in chime a made by using he esiden sa coplasmic e iculum p o ein calseques in wi h mu a ed aequo in [15]. In addi ion, we ha e also used he same low-Ca2+ a fini y aequo in, a ge ed o mi ochond ia ins ead o ER, o measu e la ge [Ca2+] ansien s in mi ochond ia a e s imula ion o ch oma fin cells [16]. The subsequen applica ion o his echnique o a wide ange o cells was only possible by using i al me hods o exp ess he aequo in chime a [17].Fig. 1 shows he plasmid o he pes simplex i us ype 1 used o clone ER- a ge ed aequo in and gene a e i uses able o exp ess he cons uc . Thanks o his echnique, i was possible o exp ess ER- a ge ed aequo in in a a ie y o cell lines (NIH3T3, PC12, GH3) and p ima y cul u es (ce ebella g anule cells, an e io pi ui a y cells and ch oma fin cells), ob aining dynamic measu emen s o [Ca2+]ER in hese cells [17,18]. METHODOLOGY Fig. 3 ou lines a ypical expe imen , which we can desc ibe as a se ies o s eps. Cell seed Cells should be seeded a 50% confluency on o glass o plas ic co e slips. A good a achmen is essen ial, as cells ha e o be pe used wi h di e en solu ions du ing he expe imen . To acili a e a achmen in some cell kinds, we ea p e iously he co e slips wi h poly-L-lysine. I is impo an o ake in o accoun ha econs i u ion o aequo in equi es p io deple ion o Ca2+o he ER (see below) by incuba ion in EGTA-con aining medium o 1–2 h. Some cells may end o de ach om glass du ing Fig. 3 Ou line o a ypical expe imen (see he ex o de ails). his pe iod. Poly-L-lysine co e ed co e slips may be ob- ained comme cially. Al e na i ely, hey may be easily p epa ed by in oducing au ocla ed co e slips in a fil- e ed s e ile 0.01 mg/ml poly-L-lysine solu ion o 5min, washing hem 2 min in s e ile wa e and finally ai d ying hem comple ely unde s e ile condi ions. Exp ession o ER- a ge ed mu a ed aequo in This s ep may be skipped i he cells come om a cell clone exp essing ER- a ge ed mu a ed aequo in (ERmu- AEQ). Fo example, we ha e s able HeLa cell clones ha exp ess his cons uc [9]. To measu e [Ca2+]ER in o he cell ypes, he ERmu AEQ plasmid should be exp essed in he desi ed cell cul u e. This can be achie ed ei he by ans ec ion o in ec ion me hods. We ha e used suc- cess ully Ca-phospha e and ans ec ion eagen s such as Lipo ec amine (Gibco Labs, G and Island, NY) and Fugene (Ho mann-La Roche, Basel, Swi ze land) o exp ess his plasmid in cell lines such as HeLa and CHO. Howe e , hese me hods a e no e ficien o exp ess he cons uc in many o he cell ypes, pa icula ly p ima y cul u es. To sol e his p oblem, we ha e used de ec i e he pes simplex i us (HSV1) ca ying he gene, and his me hod has al- lowed o ob ain a good exp ession in a se ies o cell lines and p ima y cul u e cells [17,18]. Adequa e exp ession is © 2002 Else ie Science L d. All igh s ese ed. Cell Calcium (2002) 32(5–6), 251–260 254 J Al a ez, M Mon e o usually ob ained a e 12 h using i us-based me hods, and 18–24 h using ans ec ion me hods. Ca2+-deple ion o he ER E en using mu a ed aequo in and coelen e azine n, e- cons i u ion o aequo in is impossible i he ER emains ull o Ca2+. I is, he e o e, necessa y o deple e com- ple ely he ER o Ca2+ o 1–2 h. This is achie ed by incuba ing he cells in medium con aining EGTA and a e e sible inhibi o o he ER-Ca2+-ATPase, such as 2,5-di- e -bu yl-benzohyd oquinone (BHQ) o ciclopia- zonic acid. These compounds p oduce a apid Ca2+de- ple ion o he ER (hal - ime 1–2 min) h ough some s ill unknown leak pa hways. Ca2+ eleased om he ER is hen ex uded ou o he cell by he plasma memb ane Ca2+pump. B iefly, he glass co e slip con aining he cells ha exp ess ERmu AEQ is ans e ed o a well (o a 24-well pla e) con aining 1ml o ex acellula medium (NaCl, 145 mM; KCl, 5mM; MgCl2, 1mM; glucose, 10mM; Hepes, 10 mM, pH 7.4) con aining 0.5mM EGTA. A e washing once wi h he same medium, 0.4 ml o deple- ion solu ion (ex acellula medium supplemen ed wi h 0.5 mM EGTA and 10␮M BHQ) is added o he well and le o 10 min a oom empe a u e. This ime is enough o emp y almos comple ely he ER o Ca2+. Recons i u ion wi h coelen e azine n Recons i u ion o aequo in wi h coelen e azine n (Molec- ula P obes Eu ope BV, Leiden, The Ne he lands) is much slowe han wi h wild- ype coelen e azine ( he hal - ime in i o is 5 h compa ed o 22min o he wild- ype [12]). This means ha longe imes o econs i u ion will gi e an almos linea inc ease in he o al luminescence ou pu . A e y impo an ac o is also o keep low he empe a u e o econs i u ion. Inc easing he empe a u e o 37 ◦C du ing econs i u ion dec eases by nea one o de o magni ude he o al luminescence o he sample, pe haps because o inc eased aequo in consump ion. The usual p ocedu e is as ollows: deple ion solu ion is emo ed and 0.2 ml o he same solu ion is added o he well. Then, 1 ␮l o coelen- e azine n (s ock p epa ed 200␮M in me hanol) is added and gen ly mixed. The pla e should be hen le in he da k (coelen e azine is ligh -sensi i e) a oom empe a u e o 1–2 h. Luminescence expe imen A e econs i u ion, he glass co e slip is ans e ed o he cell chambe o a home-made luminome e . Cells a e hen ini ially pe used o 5 min wi h ex acellula medium con aining 0.5 mM EGTA in o de o wash comple ely BHQ and elease he inhibi ion o he ER-Ca2+-ATPase. Then, he usual expe imen s a s by pe using ex acel- lula medium con aining 1 mM Ca2+ o efill he ER. Once [Ca2+]ER eaches he s eady-s a e, di e en kinds o expe - imen s may be pe o med (see below). I is impo an o know also ha e e y expe imen has o end wi h a final s ep o cell lysis, in o de o allow calib a ion o he lumi- nescence in o [Ca2+] alues (see below). The equipmen o luminescence measu emen s in cell popula ions is com- posed o se e al componen s ha can be easily assembled. B iefly, we use a Elec on Tubes (Ruislip, U.K.) pho omul- iplie 9789A e ige a ed a 4 ◦C inside a cool box. The 10 mm pho oca hode is placed in almos di ec con ac wi h he he mos a ized pe usion chambe (Fig. 3), and co e s mos o he su ace o he glass co e slip. A sys em o eigh elec o al es allows pe using di e en solu ions, placed in a nea by ba h, ha flow by g a i y du ing he expe imen . The pho omul iplie is usually se a 1–1.2 kV, and he ou pu goes ia an amplifie -disc imina o AD2 up o a coun e / ime PCB CT1 ca d in he com- pu e , bo h om Elec on Tubes. Luminescence da a a e s o ed e e y 50ms, a e aged e e y second and ans- o med in o [Ca2+] alues h ough a home-made so wa e (see below). Calib a ion o luminescence da a In o de o calib a e he da a ob ained in e ms o [Ca2+]ER, we need o know he o al amoun o luminescence ha can be emi ed by he sample. Fo ha , a he end o e e y expe imen i is essen ial o pe use lysis solu ion (10 mM CaCl2and 100 ␮M digi onin in wa e ). Cell pe meabi- liza ion in he p esence o excess Ca2+ apidly eleases all he emaining aequo in luminescence. To unde s and why his o al luminescence is equi ed, we mus look a he calib a ion cu es in Fig. 2. The figu e shows cal- ib a ion cu es in a ypical d awing o log L/Lmax agains pCa (−log[Ca2+]). Lis he luminescence (coun s pe sec- ond) o a sample o aequo in placed in a medium wi h a gi en [Ca2+], and Lmax is he o al luminescence ha can be eco ded om he sample a ha momen . L/Lmax is, he e o e, he ac ion o he o al luminescence eco ded om he sample in 1 s. Fo e e y [Ca2+], a pa icula ac- ion o he o al aequo in luminescence is emi ed pe second, and he calib a ion cu es show he ela ionship be ween his ac ion and he [Ca2+]. The e o e, c ude luminescence alues ha e no meaning in e ms o [Ca2+] un il hey a e ans o med in o a ios L/Lmax. The final calib a ed esul s a e he e o e independen o he o al amoun o econs i u ed aequo in in he sample. On he o he hand, as aequo in is being p og essi ely consumed, he alue o Lmax ( he o al emaining luminescence o he sample) dec eases con inuously along he expe imen s. Because o his, he beha io o aequo in luminescence is qui e di e en o ha o Ca2+-sensi i e fluo escen dyes. Cell Calcium (2002) 32(5–6), 251–260 © 2002 Else ie Science L d. All igh s ese ed. ER [Ca2+] and aequo in 255 Fo example, a cons an le el o luminescence (L) co e- sponds o a con inuous inc ease in L/Lmax a ios and hus, o a con inuous inc ease in [Ca2+]. Vice- e sa, o ge a cons an le el o [Ca2+], we need he L/Lmax a io o be- come cons an , and o his we need he luminescence o be dec easing exponen ially. C ude eco ds o luminescence a e s o ed in compu e files ha con ain da a sampled e e y 50ms. To ans o m hese da a in [Ca2+], a compu e p og am a e ages hem 20- old o ob ain a da a a e o 1s−1, sub ac s he back- g ound and calcula es he ac ions L/Lmax a e e y poin along he expe imen . Lis he luminescence alue a e - e y poin (minus he backg ound) and Lmax is he in eg al o luminescence (minus he backg ound) om ha poin o he end o he expe imen . L/Lmax alues a e hen ans- o med in o [Ca2+] alues using he ollowing ma hema i- cal algo i hm [19]. [Ca2+](in M)= a io +( a io ×KTR)−1 KR−( a io ×KR), whe e a io =L Lmax ×λ1/n . This algo i hm was de i ed om a ma hema ical model p oposed o iginally o explain om a molecula poin o iew he Ca2+-dependence o aequo in luminescence [20], bu i can be used as a simple ma hema ical ans o ma ion independen o he model. The alues o he pa ame e s o he algo i hm ha should be used o calcula e [Ca2+]in expe imen s using ERmu AEQ econs i u ed wi h coelen- e azine n a e: A 22 ◦C:KR=5×107,KTR =1.597 ×105, n=1.271,λ=0.02512, A 37 ◦C: KR=8.47 ×107,KTR =1.656 ×105, n=1.2038,λ=0.138. Rega ding he significance o he pa ame e s in he model [20], i is wo h indica ing ha nwas he numbe o Ca2+-binding si es in he model, and has a alue o nea 3 in na i e aequo in. The smalle alue o nobse ed he e is mainly due o he mu a ion o one o he Ca2+-binding si es, and co esponds wi h a dec ease in he slope o he calib a ion cu e (compa e he slope o he AEQ1 cu e wi h hose o AEQ2 o AEQ3 ones in Fig. 2). The pa am- e e λis he a e cons an o aequo in consump ion a sa u a ing [Ca2+]. This pa ame e was no included in he o iginal desc ip ion o he algo i hm [19], because he maximum a e cons an o na i e aequo in econs i u ed wi h wild- ype coelen e azine is 1.0 s−1. Recons i u ion wi h coelen e azine n educes conside ably he maximum a e cons an , and his allows eco ding high [Ca2+]ER al- ues wi h smalle aequo in consump ion, pa icula ly a 22 ◦C. MEASUREMENT OF [Ca2+]ER Fig. 4 shows a de ailed analysis o wo expe imen s pe - o med a 37 ◦C (le panels) and 22◦C ( igh panels). HeLa cells exp essing ER- a ge ed mu a ed aequo in we e e- cons i u ed wi h coelen e azine n as de ailed abo e, in o- duced in he he mos a ized chambe o he luminome e and pe used wi h ex acellula medium con aining EGTA. Then, when indica ed in he figu e, ex acellula medium con aining 1 mM Ca2+ins ead o EGTA was pe used. The uppe panels show he eco ds o luminescence (coun s pe second) ob ained wi h his p o ocol a e e y empe a- u e. A 37 ◦C (le panels), addi ion o Ca2+ o he ex acel- lula medium igge ed a apid inc ease in luminescence, which eached a peak and hen dec eased apidly down o nea backg ound le els. Mos o aequo in was consumed in his peak, because final cell lysis wi h digi onin induced only a small luminescence peak, con aining 1–2% o he o al luminescence. This is be e seen in he middle le panel, which shows he pe cen age o aequo in consump- ion along he expe imen . I is appa en ha nea ly all he luminescence o aequo in in he sample was emi ed du ing he peak, so ha only 1–2% o he o al lumines- cence emained o be emi ed a he momen o cell lysis. The [Ca2+] measu ed a ha momen , when >98% o ae- quo in had been consumed, was ela i ely low, 1–5␮M, indica ing he p esence o a small compa men in he ER con aining low [Ca2+][10]. Fig. 4 Analysis o aequo in luminescence expe imen s pe o med a 37◦C (le panels) and 22◦C ( igh panels). Uppe panels, c ude luminescence eco ds. Middle panels, aequo in consump ion along he expe imen calcula ed as he pe cen age o aequo in emaining. Lowe panels: (A) calib a ed [Ca2+] ob ained by di ec applica ion o he algo i hm; (B) calib a ed [Ca2+] ob ained a e educing he o al luminescence (Lmax)by2%. © 2002 Else ie Science L d. All igh s ese ed. Cell Calcium (2002) 32(5–6), 251–260 256 J Al a ez, M Mon e o This 1–2% o aequo in con ained in a low-Ca2+en i on- men has a d ama ic influence in he calib a ion. In he lowe le panel, cu e A shows he [Ca2+] alues ob ained by applying di ec ly he algo i hm o he expe imen al da a. Addi ion o Ca2+induces an inc ease in [Ca2+]ER, ha ises smoo hly un il nea 500␮M and hen dec eases slowly o each again e y low [Ca2+]ER alues wi hin ew minu es. To unde s and why he [Ca2+]ER dec eases in- s ead o eaching a s able s eady-s a e, we should no e fi s ha he dec ease in [Ca2+]ER s a s when less han 20% o aequo in emains able o emi ligh (do ed line). A ha momen , he 1–2% o aequo in p esen in a low-Ca2+ compa men s a s o become quan i a i ely impo an . In ac , a ew minu es la e , his low-Ca2+compa men becomes dominan when mos o aequo in has been con- sumed, and he appa en [Ca2+]ER e u ns o e y low al- ues. The a i ac in oduced by his small compa men can be o e come by assuming ha aequo in in ha com- pa men does no emi a significan amoun o ligh du - ing he ini ial pa o he expe imen . In ha case, we can educe he o al amoun o aequo in in he algo i hm by aking ou he 1–2% o aequo in o he low-Ca2+com- pa men . The exac pe cen age o his compa men can be es ima ed om he amoun o aequo in luminescence eleased by lysing he cells a he end o he expe imen , when luminescence has e u ned o nea -backg ound le - els. The esul o his ecalcula ion o he same da a, educ- ing he alue o Lmax by 2%, is shown in cu e B. We can see ha his small change in he alue o he o al lumi- nescence ha dly a ec s he ini ial inc ease in [Ca2+]ER, bu makes disappea he subsequen dec ease. Now [Ca2+]ER s abilizes a a ound 500␮M, al hough he apid consump- ion o aequo in allows moni o ing his high [Ca2+]ER alue only o ew minu es. Simila esul s a e ob ained when his p o ocol is ca ied ou a 22 ◦C, excep o he ac ha s eady-s a e [Ca2+]ER alues can be measu ed o a longe ime. Addi ion o Ca2+induced a much b oade peak o luminescence ha ended in a long ail (uppe igh panel). A he end o ha pe iod, cell lysis eleased a small amoun o ligh , usually sligh ly highe han ha ob ained in he expe imen s a 37 ◦C. This is p obably due o he o e lap o he long ail, ha is, he incomple e consump ion o he high-Ca2+ compa men . Again he e, he calib a ed [Ca2+]ER da a show a smoo h inc ease ollowed by s abiliza ion a abou 500␮M, and hen slow dec ease due o he low-Ca2+com- pa men (cu e A). The dec ease in [Ca2+]ER s a s also he e when only 20% o aequo in emains ac i e in he sample (do ed line, igh panels). Recalcula ion o he da a educing again Lmax in 2% p oduced cu e B, ha shows a s able s eady-s a e ha allows pe o ming di e en kinds o expe imen al maneu e s o mo e han 10 min. This simple expe imen cons i u es a good example o he abili y o aequo in o de ec he e ogenei ies in [Ca2+]. In his case, as aequo in consump ion in high [Ca2+] a eas allowed de ec ing a small compa men (2% o o al) wi h much lowe [Ca2+]. This compa men , whose s uc u al na u e is unknown, may ha e been esponsible o he fi s measu emen s o [Ca2+]ER wi h ER- a ge ed aequo in in he ange 1–5 ␮M[4]. Simila alues we e also ob ained when we exp essed ou ER- a ge ed cons uc con aining na i e aequo in in HeLa cells [10]. On he o he hand, his kind o expe imen s also cons i u e e idence ha mos o he ER (98% in HeLa cells) has a nea ly homogeneous ee [Ca2+] a ound 500␮M. The p esence o compa men s wi h g ossly di e en ee [Ca2+] would ha e been easily de ec able by he p esence o di e en a es o consump- ion along he efilling p ocess. Compa ing he da a ob ained a bo h empe a u es, he main eason o he slowes aequo in consump ion a 22 ◦C is ha he maximum a e o aequo in consump- ion (λ) dec eases abou fi e- old a 22 ◦C compa ed wi h ha a 37 ◦C (see also Fig. 2). This inc eases he ime ha [Ca2+]ER can be eco ded a s eady-s a e a 22 ◦C, e en hough he s eady-s a e [Ca2+]ER le el is he same a bo h empe a u es. As a consequence, while a 37 ◦C s eady-s a e [Ca2+]ER alues can be moni o ed o only 2–3 min, a 22 ◦C he measu ing ime a he same [Ca2+]ER alues becomes 15–20 min. Sol ing he p oblem o as consump ion a 37 ◦C would equi e in oducing a new mu a ion in he aequo in molecule o educe u he i s Ca2+a fini y. As men ioned abo e, he echnique can be used wi h many cell ypes, e en p ima y cul u es, p o ided ha he p epa a ion is highly pu e and ha exp ession o he ERmu AEQ gene becomes possible. The pu i y o he p epa a ion is e y impo an in p ima y cul u es because measu emen s in cell popula ions canno sepa a e he signal coming om di e en cell ypes, and he in ensi y o he exp ession may also be di e en in di e en cell ypes. We ha e measu ed [Ca2+]ER in some p epa a ions ha can be ob ained highly pu e, such as ch oma fin cells and ce ebella g anule cells. Fig. 5 shows a ypical expe imen pe o med in ch oma fin cells (see also [18]). The uppe panel shows he eco d o luminescence and he lowe one he calib a ed [Ca2+]ER alues. Addi ion o ex acellula Ca2+s a s efilling o he ER, eaching le els close o 500␮M (lowe panel). Then, a se ies o addi ions o 5 mM ca eine we e made. Each ca eine addi ion in- duced a apid bu pa ial dec ease o [Ca2+]ER, ollowed by eco e y o he same concen a ions. This maneu e was epea ed many imes, and [Ca2+]ER always eco e ed o s eady-s a e alues. This epe i i e eco e ing is ex emely impo an as a es ha aequo in consump ion does no a ec significan ly he [Ca2+]ER alues ob ained in hese cells. Finally, in he las addi ion, 50 mM ca eine was pe used, p oducing a as and ull emp ying o he ER. I is e y in e es ing o compa e he c ude luminescence Cell Calcium (2002) 32(5–6), 251–260 © 2002 Else ie Science L d. All igh s ese ed. ER [Ca2+] and aequo in 257 Fig. 5 E ec o ca eine on ER- a ge ed aequo in luminescence and calib a ed [Ca2+]ER. Bo ine ch oma in cells we e in ec ed wi h pHSVe AEQ o exp ess ER- a ge ed mu a ed aequo in. A e econs i u ion wi h coelen e azine n, e illing o he ER was s a ed by pe using ex acellula medium con aining 1mM Ca2+as indica ed. Then, se e al consecu i e s imula ions wi h 5 o 50mM ca eine we e pe o med as indica ed. The uppe panel shows he c ude luminescence eco d, and he lowe one he calib a ed [Ca2+]ER alues. Tempe a u e was 22◦C. da a wi h he calib a ed alues o unde s and he cali- b a ion o aequo in. Fi s , i is e iden ha a s eady-s a e luminescence is no indica i e o a s eady-s a e [Ca2+]. Fo example, a e he addi ion o Ca2+ o he ex acellu- la medium, luminescence eached a s eady-s a e be o e he fi s addi ion o ca eine (uppe panel), bu calib a ed [Ca2+]ER was s ill going up. Simila ly, [Ca2+]ER eco e ed o simila alues a e each s imula ion, bu luminescence dec eased con inuously along he expe imen . The echnique allows also pe o ming expe imen s wi h pe meabilized cells (Fig. 6). The p o ocol he e is iden ical o ha desc ibed abo e un il he glass co e slip is placed in he luminome e and BHQ is washed. A ha poin , in- s ead o pe using ex acellula medium con aining 1 mM Ca2+, we pe use in acellula medium con aining 0.5 mM EGTA and 100␮M digi onin o 1min (ma ked “digi onin” in he figu e). These a e he condi ions equi ed o pe - meabilize HeLa cells, bu o o he cells he concen a ion o digi onin o he ime necessa y may be di e en . Then, Fig. 6 E ec o InsP3on [Ca2+]ER in pe meabilized HeLa cells. HeLa cells exp essing ER- a ge ed aequo in we e econs i u ed wi h coelen e azine n, in oduced in he luminome e and pe used wi h 0.5mM EGTA-con aining ex acellula medium. Then, cells we e pe meabilized by pe usion o in acellula medium (NaCl, 10mM; KCl, 140mM; MgCl2, 1mM; KH2PO4, 1mM; ATP-Mg, 2mM; Hepes, 20mM, pH 7.0) con aining 0.5mM EGTA and 100␮M digi onin o 1min as indica ed. Then, he ER was e illed by pe usion o in acellula medium con aining 100nM Ca2+(bu e ed wi h EGTA). Once he s eady-s a e [Ca2+]ER had been eached, ei he 0.1 o 2␮M InsP3we e added as indica ed. Tempe a u e was 22◦C. in acellula medium con aining 100 nM Ca2+(bu e ed wi h EGTA) is pe used o efill he ER. Fig. 6 shows ha he ER is efilled a a simila a e ha in in ac cells (Fig. 4) and eaches simila s eady-s a e [Ca2+]ER le els. A ha poin , we can es he e ec o an in acellula Ca2+mobi- lize such as inosi ol 1,4,5- isphospha e (InsP3). The fig- u e shows he e ec o a maximal concen a ion o InsP3 (2 ␮M) and a submaximal one (0.1 ␮M). These expe imen s on pe meabilized cells may be pa icula ly use ul when we wan o measu e p ecisely he esponse o he ER o a ce ain non-pe meable in acellula agen . ADVANTAGES AND DISADVANTAGES OF THE TECHNIQUE In his sec ion, we will y o dissec he main p os and cons o aequo in-based me hods o measu ing [Ca2+]ER dynam- ics. On he posi i e side, we can men ion he ollowing poin s: 1. Selec i i y o a ge ing. I has been shown bo h by im- munofluo escence [9] and by elec on mic oscopy [10] ha he p obe is co ec ly sen and e ained in he ER. This is an impo an ad an age wi h espec o fluo es- cen dyes, ha canno be a ge ed specifically. 2. La ge dynamic ange and high signal- o-noise a io.The luminescence o aequo in can change by a ac o o se e al o de s o magni ude a e he inc ease in [Ca2+], and e e y ype o aequo in is able o ollow [Ca2+] changes wi hin a span o nea ly wo o de s o magni- ude o [Ca2+]. In addi ion, he a ailabili y o di e en kinds o aequo ins (Fig. 2) wi h di e en Ca2+-a fini ies allows explo ing a wide ange o [Ca2+], om 10−7 o 10−3M. © 2002 Else ie Science L d. All igh s ese ed. Cell Calcium (2002) 32(5–6), 251–260 258 J Al a ez, M Mon e o 3. S aigh o wa d calib a ion. As shown abo e, calib a- ion o luminescence in o [Ca2+] alues equi es only o know he o al luminescence o he sample. This is easily measu ed by lysing he cells in Ca2+-con aining medium a he end o he expe imen . A e ha , cal- ib a ion in [Ca2+] is ob ained immedia ely by apply- ing he algo i hm desc ibed abo e h ough a compu e p og am. The possible p esence and size o a low-Ca2+ compa men should be explo ed and co ec ed as de- sc ibed abo e, i possible. On he o he hand, aequo in is ha dly sensi i e o pH in he physiological ange, and Mg2+is unable o igge luminescence. 4. Low-cos equipmen . The equipmen necessa y o pe - o m he luminescence measu emen s is no comme - cial bu can be easily assembled om a se ies o low-cos componen s. Simila ly, he so wa e o ans o m lumi- nescence in o [Ca2+] can be eely ob ained om ou labo a o y. In he o he side o he balance, his me hodology has also some impo an d awbacks ha should be conside ed: 1. Small amoun o ligh . The amoun o emi ed ligh is ela i ely small, and his makes di ficul pe o ming single-cell imaging s udies. Recen ly, howe e , i has been shown ha he use o a high-sensi i i y came a combined wi h he high gene exp ession p o ided by i uses, allows making single-cell s udies o mi ochon- d ial o nuclea [Ca2+] using a ge ed aequo in [21,22]. Ne e heless, he applica ion o his echnique o he measu e o [Ca2+]ER is mo e p oblema ic, because o he di e en pa e n o [Ca2+] changes in he ER com- pa ed wi h mi ochond ia, nucleus o cy osol. In he ER, he s eady-s a e [Ca2+]ER is e y high, and we ha e o educe as much as possible he a e o emission o luminescence (using he lowes Ca2+-a fini y aequo in) in o de o be able o moni o ha s eady-s a e o some ime. In addi ion, changes o ha s eady-s a e usually consis in a dec ease o [Ca2+]ER. In e ms o luminescence, his means ha we ha e a low le el o luminescence in s eady-s a e, and should y o de ec a dec ease on ha small le el. In con as , aequo in is much be e designed o ollow [Ca2+] changes in o ganelles such as mi ochond ia o nucleus, whe e he es ing [Ca2+] is low and he esponse o s imula- ion consis s in a la ge bu ansien inc ease o e he es ing le els. 2. Deple ion o Ca2+o he ER. As men ioned abo e, e- cons i u ion o aequo in wi h coelen e azine equi es deple ing he ER o Ca2+ o 1–2 h. The ques ion he e is whe he o no his p olonged deple ion al- e s somehow he cell [Ca2+]ER esponses ha a e going o be s udied. This is an impo an ques ion, because he ac i i y o many chape one p o eins in he ER depends on [Ca2+]ER, and deple ion o Ca2+ o he ER causes an accumula ion o un olded p o- eins in he ER lumen, which leads o he ac i a ion o wo highly conse ed s ess esponses, he ER o e - load esponse and he un olded p o ein esponse [23]. These esponses lead o ac i a ion o he exp ession o a se ies o genes oge he wi h an inhibi ion o he ini ia ion o p o ein syn hesis, ha may end by causing cell inju y o e en apop osis. We ha e o say, howe e , ha mos o hese phenomena ha e been s udied in cells subjec ed o ER-Ca2+-deple ion bu kep in a Ca2+-con aining ex acellula medium. Unde hese condi ions, ER-Ca2+-deple ion coexis s wi h an inc eased cy osolic [Ca2+]. Ins ead, du ing ae- quo in econs i u ion, he ex acellula medium con- ains EGTA and cy osolic [Ca2+] is low. This di e ence has been shown o be e y impo an ega ding ER s uc u e. While ER-Ca2+-deple ion in Ca2+-con aining medium led o agmen a ion and esicula iza ion o he ER, he e was no change in ER s uc u e when ER-Ca2+-deple ion was ca ied ou in he absence o ex acellula Ca2+[24]. On he o he hand, he s eady- s a e [Ca2+]ER le els measu ed wi h aequo in a e sim- ila o hose measu ed wi h cameleons [25] o wi h fluo escen dyes [26–28], in he ange 300–500 ␮M, sugges ing ha ER-Ca2+-deple ion does no a ec he basic [Ca2+]ER homeos a ic machine y. In ou expe i- ence, mo eo e , we ha e no ound significan di e - ences in he [Ca2+]ER esponses by a ying he pe iod o deple ion om 1 o 4 h. O cou se, we canno exclude ha some cellula esponses may become modified a e his ea men . 3. High a e o consump ion. The leng h o he expe i- men s is conside ably limi ed by he apid consump ion o aequo in ha s a s immedia ely a e ein oduc- ion o Ca2+.Fig. 4 shows ha , a 37 ◦C, nea ly 90% o aequo in has been al eady consumed when he s eady-s a e is eached. This means ha he e is ha dly any ime le o es he e ec s o agonis s o o he pos- sible expe imen al maneu e s. This is why we pe o m mos o ou [Ca2+]ER expe imen s a 22 ◦C. A his em- pe a u e, he s eady-s a e is eached when aequo in consump ion is s ill a ound 60% (Fig. 4), and eason- able measu emen s can be s ill ob ained du ing 10–20 mo e minu es (Fig. 5). The p oblem o ime-limi a ion may be sol ed by educing u he he Ca2+-a fini y o aequo in, pe haps by in oducing a new mu a ion in any o he Ca2+-binding si es, al hough his modifica- ion will p obably educe conside ably he ligh ou pu in s eady-s a e. 4. DNA exp ession me hod equi ed. T ans ec ion o in ec- ion me hods a e equi ed o exp ess he ER- a ge ed aequo in in he cell ype o in e es . The e ficiency o hese me hods is a iable and depends on he cell ype. In addi ion, exp ession o he p o ein equi es a leas Cell Calcium (2002) 32(5–6), 251–260 © 2002 Else ie Science L d. All igh s ese ed. ER [Ca2+] and aequo in 259 12 h o i us-based me hods and 18–24 h o ans- ec ion me hods. This means ha p ima y cul u e cells canno be s udied immedia ely a e ex ac ion and ha e o be cul u ed, seeded and ans ec ed/in ec ed. Finally, cell in ec ion may ha e dele e ious e ec s. The e o e, es s o cell unc ionali y a e in ec ion should be ca ied ou in pa allel o show ha in ec ion does no modi y o he pa ame e s o cell unc ion. CONCLUSION Aequo in-based me hods o measu e [Ca2+]ER combine a high specifici y o a ge ing wi h a la ge dynamic ange o [Ca2+] measu emen om mic omola o millimola le - els and also a high signal- o-noise a io. In con as , hey keep se e e limi a ions pa icula ly in e ms o in ensi y o ligh emission and apid consump ion o he p obe. The balance be ween p os and cons will de e mine in which kind o applica ions i may be mo e adequa e. I may be also use ul o combine measu emen s o [Ca2+]ER wi h ER- a ge ed aequo in and o he me hodologies. Fluo es- cen dyes, o example, a e much be e o do single-cell imaging and ha e no p oblems o consump ion, bu lack specifici y o a ge ing, ha e a smalle dynamic ange and signal- o-noise a io, and calib a ion is complica ed due o in e e ence om Mg2+, pH o jus non-specific dis ibu- ion o he dye. 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