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The Jou nal o Cell Biology, Volume 144, Numbe 2, Janua y 25, 1999 241–254
h p://www.jcb.o g 241
Ca
2
1
-induced Ca
2
1
Release in Ch oma in Cells Seen om
inside he ER wi h Ta ge ed Aequo in
Ma ia Te esa Alonso,* Ma ia José Ba e o,* Ped o Michelena,
‡
Es ela Ca nice o,* Inmaculada Cuchillo,
‡
An onio G. Ga cía,
‡
Ja ie Ga cía-Sancho,* May e Mon e o,* and Ja ie Al a ez*
*Ins i u o de Biología y Gené ica Molecula , Depa amen o de Bioquímica y Biología Molecula y Fisiología, Facul ad de
Medicina, Uni e sidad de Valladolid y Consejo Supe io de In es igaciones Cien í icas, E-47005 Valladolid, Spain; and
‡
Ins i u o de Fa macología Teó ilo He nando, Depa amen o de Fa macología y Te apéu ica, Facul ad de Medicina,
Uni e sidad Au ónoma de Mad id, E-28029 Mad id, Spain
Abs ac .
The p esence and physiological ole o Ca
2
1
-
induced Ca
2
1
elease (CICR) in nonmuscle exci able
cells has been in es iga ed only indi ec ly h ough mea-
su emen s o cy osolic [Ca
2
1
] ([Ca
2
1
]
c
). Using a ge ed
aequo in, we ha e di ec ly moni o ed [Ca
2
1
] changes
inside he ER ([Ca
2
1
]
ER
) in bo ine ad enal ch oma in
cells. Ca
2
1
en y induced by cell depola iza ion ig-
ge ed a ansien Ca
2
1
elease om he ER ha was
highly dependen on [Ca
2
1
]
ER
and sensi ized by low
concen a ions o ca eine. Ca eine-induced Ca
2
1
e-
lease was quan al in na u e due o modula ion by
[Ca
2
1
]
ER
. Whe eas ca eine eleased essen ially all he
Ca
2
1
om he ER, inosi ol 1,4,5- isphospha e (InsP
3
)-
p oducing agonis s eleased only 60–80%. Bo h InsP
3
and ca eine emp ied comple ely he ER in digi onin-
pe meabilized cells whe eas cyclic ADP- ibose had no
e ec . Ryanodine induced pe manen emp ying o he
Ca
2
1
s o es in a use-dependen manne a e ac i a-
ion by ca eine. Fas con ocal [Ca
2
1
]
c
measu emen s
showed ha he wa e o [Ca
2
1
]
c
induced by 100-ms de-
pola izing pulses in ol age-clamped cells was delayed
and educed in in ensi y in yanodine- ea ed cells. Ou
esul s indica e ha he ER o ch oma in cells beha es
mos ly as a single homogeneous hapsiga gin-sensi i e
Ca
2
1
pool ha can elease Ca
2
1
bo h ia InsP
3
ecep-
o s o CICR.
Key wo ds: endoplasmic e iculum • aequo in • ch o-
ma in cells • calcium • yanodine
A
p esen , he ole played by yanodine ecep o s
(RyR)
1
in he homeos asis o in acellula Ca
2
1
in
nonmuscle cells is unclea . Mammalian issues ex-
p ess h ee iso o ms, RyR1, RyR2, and RyR3, encoded by
di e en genes. RyR1 and RyR2 a e exp essed p edomi-
nan ly in sa coplasmic e iculum o skele al muscle and
hea , espec i ely, whe e hey ha e an essen ial ole o
igge muscle con ac ion (Su ko and Ai ey, 1996; Zucchi
and Ronca-Tes oni, 1997). RyR3 was o iginally iden i ied
in b ain (Hakama a e al., 1992), bu in ac all h ee iso-
o ms a e ac ually exp essed in b ain, and he majo b ain
iso o m appea s o be RyR2 (McPhe son and Campbell,
1993; So en ino and Volpe, 1993; Giannini e al., 1995).
RyRs a e widely dis ibu ed in many o he di e en is-
sues (Giannini e al., 1995; Mack ill e al., 1997), including
he ad enal gland, and hey a e in many cases coexp essed
wi h one o mo e iso o ms o inosi ol 1,4,5- isphospha e
ecep o s (InsP
3
R) (Wal on e al., 1991; Poulsen e al.,
1995).
The easons o he p esence o mul iple Ca
2
1
elease
mechanisms in he same cell a e no clea ( o discussion
see New on e al., 1994; Su ko and Ai ey, 1996). A possi-
ble eason o he coexis ence o bo h InsP
3
R and RyR in
he same cells could be ha hey migh elease Ca
2
1
om
di e en compa men s, and wi h a di e en physiological
signi icance. They may be modula ed by di e en second
messenge s, such as InsP
3
in he case o InsP
3
R o cyclic
adenosine diphospha e ibose (cADPR) o he RyR (Lee,
1998). Howe e , al hough InsP
3
is a well-es ablished phys-
iological ac i a o o he InsP
3
R, he ole o cADPR as ac-
i a o o modula o o RyR in he p esence o physiologi-
M.T. Alonso and M.J. Ba e o con ibu ed equally o his wo k.
Add ess co espondence o J. Al a ez, Depa amen o de Bioquímica y
Biología Molecula y Fisiología, Facul ad de Medicina, Ramón y Cajal 7,
E-47005 Valladolid, Spain. Tel: (34) 983-423085. Fax: (34) 983-423588.
E-mail: [email p o ec ed]
1.
Abb e ia ions used in his pape :
[Ca
2
1
]
c
, cy osolic [Ca
2
1
]; [Ca
2
1
]
ER
, ER
[Ca
2
1
]; BAPTA, 1,2-bis(2-aminophenoxy)e hane-N,N,N
9
,N
9
- e aace ic
acid; cADPR, cyclic adenosine diphospha e ibose; CICR, Ca
2
1
-induced
Ca
2
1
elease; CPA, ciclopiazonic acid; DMPP, 1,1-dime hyl-4-phenyl-
pipe azinium iodide; HSV-1, he pes simplex i us ype 1; InsP
3
R, InsP
3
e-
cep o ; InsP
3
, inosi ol 1,4,5- isphospha e; i u, in ec ious i us uni s; RyR,
yanodine ecep o .
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The Jou nal o Cell Biology, Volume 144, 1999 242
cal concen a ions o ATP emains con o e sial (Su ko
and Ai ey, 1996; Zucchi and Ronca-Tes oni, 1997). In neu-
onal cells, RyR could be ac i a ed ia he classical Ca
2
1
-
induced Ca
2
1
elease (CICR) mechanism a e Ca
2
1
en y
h ough ol age-dependen Ca
2
1
channels. S udies in some
neu onal p epa a ions ha e shown ha depola izing s im-
uli p oduce an inc ease in cy osolic [Ca
2
1
] ([Ca
2
1
]
c
) ha
may be due in pa o Ca
2
1
elease om in acellula
s o es (Ve kh a sky and Shmigol, 1996). Howe e , li le di-
ec e idence has been p esen ed o CICR in nonmuscle
cells; his is due o he di icul ies in sepa a ing he con ibu-
ion o Ca
2
1
en y and Ca
2
1
elease o he [Ca
2
1
]
c
signal and
o he nonspeci ici y o ca eine and o he pha macological
agen s used o ac i a e RyR.
To s udy CICR in neu onal cells, i would be g ea ly ad-
an ageous o measu e [Ca
2
1
] speci ically inside he Ca
2
1
s o es. We ha e ecen ly epo ed a me hod o measu e
[Ca
2
1
] in he lumen o he endoplasmic e iculum ([Ca
2
1
]
ER
)
o in ac cells, he main in acellula Ca
2
1
s o e, by using an
ER- a ge ed aequo in (Mon e o e al., 1995, 1997a,b; Ba -
e o e al., 1997), which can be exp essed in di e en ypes
o cells using a i al ec o (Alonso e al., 1998). This ech-
nique is ideal o s udy di ec ly CICR, because Ca
2
1
elease
can be measu ed independen ly o he a ia ions in [Ca
2
1
]
c
.
He e we ha e used his echnique o moni o [Ca
2
1
]
ER
in
ch oma in cells. These neu oendoc ine cells ha e a po en
ca eine-sensi i e Ca
2
1
elease mechanism (Cheek e al.,
1990), and Ca
2
1
en y h ough se e al ypes o ol age-
dependen Ca
2
1
channels can be induced by K
1
depola iza-
ion o mo e physiologically, using nico inic agonis s (Núñez
e al., 1995; La a e al., 1998). Di ec e idence o a CICR
mechanism wo king unde physiological condi ions in
hese cells has no been p o ided. Howe e , we ha e e-
po ed ecen ly ha he ca eine-sensi i e Ca
2
1
s o es may
modula e ca echolamine sec e ion induced by depola iza-
ion wi h high K
1
in hese cells. Ca echolamine sec e ion
was educed a e s o e emp ying wi h ca eine, and eco -
e ed as Ca
2
1
s o es e illed du ing consecu i e K
1
/Ca
2
1
pulses. The main conclusion om ha wo k was ha Ca
2
1
s o es could ha e a double ole, ac ing ei he as a sink o as
a sou ce o Ca
2
1
, depending o hei s a e o illing (La a e
al., 1997).
Addi ionally, se e al ques ions ega ding he unc ion o
RyR in ch oma in cells emain unanswe ed. Fo ins ance,
ch oma in cells ha e also InsP
3
R, which a e a leas in
pa colocalized wi h RyR2 in he ER (Poulsen e al.,
1995). The p esence o sepa a e o o e lapping Ca
2
1
pools
esponsi e o ei he InsP
3
, ca eine, o cADPR, hei di -
e en ial sensi i i y o inhibi o s o he ER Ca
2
1
-pump
such as hapsiga gin, and he physiological signi icance o
he di e en Ca
2
1
elease mechanisms, has been a subjec
o deba e o many yea s (Cheek e al., 1991; Liu e al.,
1991; Robinson and Bu goyne, 1991; S aude man e al.,
1991; Mo i a e al., 1997). On he o he hand, he mecha-
nism o Ca
2
1
elease induced by ca eine is qui e pa icula
because inc easing concen a ions o ca eine elease Ca
2
1
in a quan al manne , a phenomenon ha has been sug-
ges ed o indica e ha he ca eine-sensi i e Ca
2
1
pool is
composed o unc ionally disc e e s o es wi h he e oge-
neous sensi i i ies o ca eine (Cheek e al., 1993, 1994a).
He e we ha e moni o ed [Ca
2
1
]
ER
in ch oma in cells o
in es iga e he mechanism o quan al Ca
2
1
elease by ca -
eine, i s ela ionship in e ms o Ca
2
1
pools wi h InsP
3
-
media ed Ca
2
1
elease, and he p esence o CICR ig-
ge ed by Ca
2
1
en y. In b ie , ou esul s indica e ha he
ER Ca
2
1
pools esponding o ca eine and InsP
3
mos ly
o e lap. The esponse o ca eine was also quan al when
s udied om inside he ER, bu his quan al esponse
could be explained by he con ol o ca eine-induced Ca
2
1
elease by [Ca
2
1
]
ER
, wi h no need o sepa a e ER com-
pa men s wi h he e ogeneous sensi i i ies o ca eine, as
p oposed p e iously (Cheek e al., 1993, 1994a). We show
ha CICR can be induced by Ca
2
1
en y elici ed ei he by
high K
1
depola iza ion o by s imula ion wi h nico inic ag-
onis s. This is consis en wi h ou p e iously p oposed
model o he Ca
2
1
s o e as a modula o o sec e ion (La a
e al., 1997). Addi ionally, using as con ocal [Ca21] mea-
su emen s, we show ha CICR pa icipa es in he gene a-
ion and p opaga ion o he Ca21 wa e induced by cell de-
pola iza ion.
Ma e ials and Me hods
P epa a ion and Cul u e o Bo ine Ch oma in Cells
Bo ine ad enal medulla ch oma in cells we e isola ed ollowing s anda d
me hods (Li e , 1984) wi h some modi ica ions (Mo o e al., 1990). Cells
we e suspended in Dulbecco’s modi ied Eagle’s medium (DME) supple-
men ed wi h 5% e al cal se um, 10 mM cy osine a abinoside, 10 mM luo-
odeoxyu idine, 50 IU ml21 penicillin and 50 IU ml21 s ep omycin. Fo
sec e ion expe imen s, cells we e pla ed in 5-cm-diam Pe i dishes (5 3
106 cells pe 5 ml o DME). Fo aequo in expe imen s, cells we e pla ed
on 12- o 13-mm glass poly-d-lysine–coa ed co e slips (0.5 3 106 cells pe
1 ml o DME). Fo measu emen s o [Ca21]c ansien s by con ocal mi-
c oscopy and ionic cu en s, cells we e pla ed on 2.5-cm-diam glass co e -
slips a a densi y o 5 3 104 cells pe ml. Cul u es we e main ained a 378C
in a humidi ied a mosphe e o 5% CO2.
P epa a ion o Vi al S ock and In ec ion o Cul u es
Cons uc ion, packaging, and i e ing o he pHSVe AEQ amplicon ec-
o and exp ession in ch oma in cells has been p e iously desc ibed
(Alonso e al., 1998). The EcoRI agmen o he e AEQmu cDNA was
subcloned in o he pHSVpuc ec o o gene a e he pHSVe AEQ. As a
helpe i us, he he pes simplex i us ype 1 (HSV-1) IE2 dele ion mu an
5dl1.2 was used wi h a i e o 2 3 107 in ec ious i us uni s (i u)/ml (Lim
e al., 1996). Ti e s o i al s ocks we e de e mined by immunocy ochemis-
y on PC12 cells. In ec ed cells we e isualized by using a abbi an i–
HSV-1 pa icle an ibody (1:10,000 dilu ion; Dako) o a mouse an i-HA1
p ima y an ibody (1:200 dilu ion; Boeh inge Mannheim) ollowed by an
alkaline phospha ase-conjuga ed an i-mouse IgG an ibody (1:200 dilu ion;
Sigma). The i e s o he ec o s ock we e 1.1 3 106 i u/ml pHSVe AEQ
and 4.1 3 106 i u/ml 5dl1.2. Ch oma in cell cul u es (5 3 105 cells/0.5 ml)
we e ou inely in ec ed wi h 1.2 3 104 i u 1 d be o e measu emen s. The
pe cen age o cells exp essing ER- a ge ed aequo in was usually z20%.
Immuno luo escence e ealed a ypical nonnuclea e icula pa e n (da a
no shown), simila o ha p e iously seen in HeLa cells (Mon e o e al.,
1995). This pa e n was no modi ied by he 1-h pe iod o ER Ca21 deple-
ion equi ed o ER aequo in measu emen s.
Measu emen s o [Ca2
1
]ER wi h Aequo in
Fo [Ca21]ER s udies, cells we e in ec ed a e 1 d in cul u e wi h HSV-1
ca ying he ER- a ge ed aequo in cons uc as desc ibed p e iously
(Alonso e al., 1998). Measu emen s o [Ca21]ER we e s a ed z16 h a e
in ec ion ha we e equi ed o allow adequa e exp ession o he a ge ed
pho op o ein. Aequo in pho oluminescence measu emen s we e pe -
o med essen ially as p e iously desc ibed (Ba e o e al., 1997). In b ie ,
cells we e deple ed o Ca21 by incuba ion o 5–10 min a 378C wi h he
sa co-endoplasmic e iculum Ca21 ATPase (SERCA) inhibi o 2,5-di- e -
bu yl-benzohyd oquinone (BHQ) 10 mM in s anda d medium con aining
145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM Hepes,
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Alonso e al. Ca2
1
-induced Ca2
1
Release Seen om he ER 243
pH 7.4, supplemen ed wi h 3 mM EGTA. Cells we e hen incuba ed o
1 h a oom empe a u e in s anda d medium con aining 0.5 mM EGTA,
10 mM BHQ, and 1 mM coelen e azine n. The co e slip was hen placed in
he pe usion chambe o a pu pose-buil he mos a ized luminome e
and s anda d medium con aining 1 mM Ca21 was pe used o e ill he ER
wi h Ca21. Measu emen s we e pe o med a 228C and [Ca21]ER alues
we e calcula ed om he luminescence eco ds using a compu e algo-
i hm (B ini e al., 1995) which ollows he calib a ion cu e epo ed be-
o e (Ba e o e al., 1997). In he expe imen s ca ied ou wi h pe meabi-
lized cells, cells we e placed in he luminome e as desc ibed abo e and
pe used o 1 min wi h in acellula -like medium (10 mM NaCl, 140 mM
KCl, 1 mM MgCl2, 1 mM KH2PO4, 2 mM ATP, 20 mM Hepes, pH 7) con-
aining 2 mM EGTA and 20 mM digi onin. Then, in acellula medium
wi hou digi onin and con aining 100 nM EGTA-bu e ed Ca21 was pe -
used o 3–5 min o e ill he ER wi h Ca21. The o al numbe o coun s
ob ained anged be ween 0.3 and 2 million.
Measu emen s o Single-cell [Ca2
1
]c
Single-cell measu emen s o [Ca21]c we e pe o med a oom empe a u e
in u a-2–loaded cells as desc ibed p e iously (Núñez e al., 1995). Cells
we e epi-illumina ed al e na i ely a 340 and 380 nm and ligh emi ed
abo e 520 nm was eco ded by an ex ended ISIS-M came a (Pho onic Sci-
ence) and analyzed using an Applied Imaging Magical image p ocesso
(Sunde land). 16 ames exci ed a e e y wa eleng h we e a e aged by
ha dwa e, wi h a ime esolu ion o z7 s o each pai o images, and
[Ca21]c was es ima ed om he a io F340/F380 by compa ison wi h u a-2
s anda ds. Field elec ic s imula ion (McIlwain and Rodnigh , 1962) was
pe o med h ough a pai o sil e elec odes placed 7 mm apa and 1.5 mm
abo e he cells. Al e na ing posi i e-nega i e squa e pulses o 50-ms du a-
ion and 60-V in ensi y we e applied a 10 Hz. The peak cu en was 100 mA.
Con ocal [Ca2
1
]c Measu emen s and
Elec ophysiological Reco dings
Elec ical measu emen s and [Ca21]c we e eco ded by using he whole-
cell pa ch-clamp echnique (Hamill e al., 1981) in combina ion wi h luo-3
based mic o luo ome y. Cells we e placed in an expe imen al chambe
ha was moun ed on he s age o an in e ed mic oscope (Diapho 200;
Nikon). Cells we e loaded ia he pa ch pipe e wi h he pen aammonium
sal o m o he luo escen dye luo-3 (100 mM). The dye was exci ed wi h
a K -A lase ligh a 488 nm and emission was de ec ed a 522 nm (32-nm
band wid h). Cells we e dialyzed wi h an in acellula solu ion con aining
135 mM CsCl2, 8 mM NaCl; 1 mM MgCl2, 20 mM Hepes, 2 mM ATP, and
0.3 mM GTP, pH 7.3. The chambe was con inuously pe used wi h
K ebs-Hepes medium. Line-scan images (0.33-mm wid h) o he in acel-
lula Ca21 dis ibu ion we e acqui ed e e y 2 ms wi h a con ocal mic o-
scope (MRC 1024; Bio-Rad), using an oil imme sion, planapoch oma ic
603 objec i e (NA 5 1.4 [Nikon]). Changes in [Ca21]c we e in e ed om
he in ensi y o luo-3 luo escence no malized o ha in es ing condi-
ions (F/F0). Whole-cell cu en s we e moni o ed wi h a DAGAN PC-
ONE pa ch-clamp ampli ie . Da a we e eco ded and analyzed wi h Igo
P o 3.02 (Wa e Me ics).
Chemicals
Coelen e azine n, u a-2AM, and luo-3 we e ob ained om Molecula
P obes. InsP3 was om Resea ch Biochemicals In e na ional. cADPR was
ob ained om Sigma and om Calbiochem-No abiochem. O he e-
agen s we e o he highes quali y a ailable om Sigma o Me ck.
Resul s
O e lap be ween Ca eine-, Ryanodine-, His amine-,
and Thapsiga gin-sensi i e Componen s o [Ca2
1
]ER
A e aequo in econs i u ion wi h coelen e azine, wi h
he ER comple ely deple ed o Ca21, he expe imen s
we e s a ed by pe using he cells wi h medium con ain-
ing 1 mM Ca21 o e ill he ER (Fig. 1 a). As in o he cells
s udied p e iously (Mon e o e al., 1995, 1997a; Ba e o e
al., 1997; Alonso e al., 1998), ull e illing o he ER e-
qui ed 3–5 min and he s eady-s a e [Ca21]ER eached was
500–800 mM (Fig. 1 a). Addi ion o his amine p oduced a
apid bu pa ial (60–80%) Ca21 emp ying o he ER, a
new [Ca21]ER s eady-s a e being eached a z200–300 mM.
Subsequen addi ion o ca eine (50 mM) induced a u -
he emp ying o nea backg ound aequo in luminescence.
The e ec s we e e e sible by washing, his allowing e ill-
ing o he ER ha was comple ed wi hin 3–5 min. Addi-
ion o ca eine a ha poin , when he s o es we e com-
ple ely e illed, igge ed a apid and comple e emp ying
o he ER. His amine was hen unable o p oduce any u -
he e ec . These esul s sugges ha essen ially he whole
ER Ca21 pool is sensi i e o ca eine and a la ge pa o i
is also sensi i e o InsP3 p oducing agonis s such as his a-
Figu e 1. E ec s o his amine and ca eine and p e ea men
wi h yanodine o hapsiga gin on [Ca21]ER. HSV-1–in ec ed
ch oma in cells we e deple ed o Ca21 and econs i u ed wi h
coelen e azine n. (a) The ER was e illed by incuba ion wi h me-
dium con aining 1 mM Ca21, hen ei he 10 mM his amine o
50 mM ca eine we e pe used as indica ed. (b) Whe e indica ed,
yanodine-p e ea ed cells we e ea ed be o e aequo in econ-
s i u ion wi h medium con aining 50 mM ca eine and 10 mM
yanodine o 2 min. Cells we e hen washed and he same ea -
men was epea ed ou imes a 2-min in e als. Whe e indica ed,
hapsiga gin-p e ea ed cells we e incuba ed wi h 1 mM hapsiga -
gin o 10 min be o e s a ing he eco d. Du ing he expe imen s,
medium con aining 1 mM Ca21 and ei he 50 mM ca eine, 1 mM
b adykinin, o 10 mM his amine was pe used as indica ed.
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The Jou nal o Cell Biology, Volume 144, 1999 244
mine. Simila esul s we e ob ained using 1 mM b adykinin
ins ead o his amine (da a no shown).
To ob ain mo e in o ma ion abou he na u e o he ca -
eine-sensi i e Ca21 pool, we s udied he abili y o he ER
o e ill in he p esence o ca eine, yanodine, o he Ca21-
ATPase inhibi o hapsiga gin. Fig. 1 b, le panel shows
ha when Ca21-deple ed cells we e incuba ed wi h Ca21-
con aining medium bu in he p esence o 50 mM ca eine,
e illing was almos abolished un il ca eine was washed
away. The small inc ease obse ed in [Ca21]ER in he p es-
ence o ca eine was insensi i e o his amine. Ryanodine
has been epo ed o lock open i e e sibly he RyR in a
use-dependen manne , ha is, when yanodine is p esen
while he channels ha e been opened by ca eine (Eh lich
e al., 1994). I all he ER had unc ional ca eine and ya-
nodine-sensi i e RyR, we would hen expec ha p e ea -
men o he cells wi h ca eine and yanodine would inhibi
also e illing o he ER wi h Ca21. Fig. 1 b, middle panel,
shows ha his is he case. Cells we e ea ed wi h i e
pulses o 50 mM ca eine and 10 mM yanodine, and hen
he d ugs we e emo ed be o e aequo in econs i u ion.
Addi ion o 1 mM Ca21 o hese cells p oduced only an
small inc ease in [Ca21]ER, ha was li le sensi i e o ca -
eine o b adykinin. The same esul s we e ob ained (Fig.
1 b, igh panel) i he cells we e p e ea ed wi h he
SERCA inhibi o hapsiga gin (1 mM). Simila e ec s
we e ob ained using lowe (20 nM) hapsiga gin concen-
a ions (da a no shown). The e o e, he whole ER has
ca eine- and yanodine-sensi i e RyRs, and e ills wi h
Ca21 ia hapsiga gin-sensi i e Ca21 pumps.
Fig. 2 illus a es he ime cou se o he use-dependen
e ec o yanodine on RyR. Fig. 2 a shows ha consecu-
i e addi ions o 50 mM ca eine p oduced compa able de-
c eases in [Ca21]ER i an in e al o 3–5 min was le be-
ween wo consecu i e addi ions o allow e illing wi h
Ca21 o he ER. I 10 mM yanodine was p esen du ing
he ca eine pulses (Fig. 2 b), he i s pulse was iden ical
o he con ol bu hen he ER became p og essi ely un-
able o e ill. A e ou pulses, he ER emained a nea
backg ound [Ca21]ER le els, and only he inc ease in
[Ca21]c elici ed by depola iza ion wi h 70 mM K1 was able
o ac i a e he Ca21 pump and p oduce a small and an-
sien inc ease in [Ca21]ER. The e ec o ca eine was no
inhibi ed by incuba ion wi h 20 mM dan olene (da a no
shown), an inhibi o o RyR ha is pa icula ly e ec i e
on he skele al muscle RyR1 (Van Winkle, 1976).
Quan al E ec o Ca eine
The e ec o ca eine has been epo ed o be quan al
(Cheek e al., 1993, 1994a), meaning ha low ca eine con-
cen a ions elease only pa o he ca eine-sensi i e pool.
The same phenomenon was obse ed he e. Fig. 3 a shows
ha addi ion o submaximal ca eine concen a ions in-
duced a apid bu pa ial emp ying o he ER, leading
wi hin 30 s o a new lowe s eady-s a e o [Ca21]ER. A ha
poin , only he addi ion o a highe ca eine concen a ion
was able o p oduce u he emp ying o he ER. Simila
quan al e ec s we e also obse ed du ing e illing o he
ER when i was ca ied ou in he p esence o ca eine.
Fig. 3 b shows ha he ER did no e ill in he p esence o
50 mM ca eine, bu e illed abou hal way once he ca -
eine concen a ion was d opped o 5 mM, and comple ely
when ca eine was washed away. Subsequen addi ion o 5
and 50 mM ca eine eleased Ca21 and eached he same
[Ca21]ER le els ob ained du ing e illing in he p esence o
hese ca eine concen a ions. The deg ee o emp ying in-
duced by a pa icula ca eine concen a ion was qui e e-
p oducible in consecu i e addi ions. Fig 3 c shows ha
consecu i e addi ions o 5 mM ca eine p oduced always
z50% emp ying o he ER, and only he addi ion o a
highe ca eine concen a ion was able o p oduce u he
emp ying. Fig. 3 d shows he e ec o yanodine added in
he p esence o a submaximal dose o ca eine. We can see
ha he i s pulses we e iden ical o he con ol, bu again
he e he ER e illed p og essi ely mo e slowly a e each
new ca eine addi ion. In his case, in con as o he ex-
pe imen shown in Fig. 2, inally a [Ca21]ER s eady-s a e
co esponding o abou hal - illing was eached, in ac , he
same [Ca21]ER ob ained ini ially a e addi ion o 5 mM
ca eine. Once a his poin , addi ion o a maximal dose o
ca eine was equi ed o induce emp ying o he emaining
po ion o he ER.
The expe imen s o Fig. 3 a e consis en wi h p e ious
esul s ob ained looking a [Ca21]c in he same cell p epa-
Figu e 2. Use-dependen inhibi ion o ER e illing by yanodine.
The ER was e illed by pe using wi h medium con aining 1 mM
Ca21. Then, se e al consecu i e s imula ions wi h 50 mM ca -
eine we e pe o med as indica ed, ei he in he absence (a) o in
he p esence (b) o 10 mM yanodine. In b, s anda d medium con-
aining 70 mM KCl ( eplacing an equimola amoun o NaCl)
was pe used when indica ed (K1). O he de ails a e as in Fig. 1.
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a ion by Cheek e al., 1994a. Those expe imen s led he
au ho s o sugges ha he e should be di e en compa -
men s wi hin he ER ha ing di e en sensi i i ies o ca -
eine. This hypo hesis would explain why yanodine only
emp ies he pool sensi i e o 5 mM ca eine bu lea es he
es o he pool un ouched, which could be only eleased
wi h a highe ca eine dose. Howe e , he e is an al e na-
i e explana ion o hese esul s, based on he egula ion
o ca eine-induced Ca21 elease by he lumenal [Ca21]. In
his hypo hesis, he esul s can be explained wi h only one
ER compa men i we assume ha submaximal ca eine
concen a ions can only elease Ca21 un il [Ca21]ER is e-
duced o a ce ain le el. The highe he ca eine concen a-
ion, he lowe he [Ca21]ER le el a ained. Bo h al e na-
i e hypo heses lead o di e en p edic ions unde some
expe imen al condi ions. In pa icula , i we ob ain hal -
illed s o es by di e en p ocedu es, e.g., by emp ying
hem wi h an agonis ac ing ia InsP3 p oduc ion o by e-
illing he s o es only hal way, he hypo hesis o se -
e al compa men s p edic s ha we should ha e all o
hem hal - illed. The e o e, a submaximal dose o ca eine
should s ill elease Ca21 om hal o hem. On he con-
a y, i he elease was di ec ly con olled by [Ca21]ER, we
would expec he e ec o 5 mM ca eine o be indepen-
den o he p ocedu e used o each ha hal - illing. The
expe imen s shown in Fig. 4 indica e ha he las hypo he-
sis is he co ec one. In Fig. 4 a, hal - illing was ob ained
by emp ying he ER wi h his amine. A e ha , 5 mM ca -
eine had no e ec . Ins ead, i 5 mM ca eine was added
wi h he ER ull o Ca21, i was able o emp y i exac ly
down o he same poin . Fig. 4 b shows he e ec o hal -
illing he ER by educing he ime o e illing. Again, he
e ec o 5 mM ca eine was s ic ly dependen on he le el
o [Ca21]ER eached a he poin i was added. I had no e -
ec a hal - illing, bu eleased 50% o he pool when he
Ca21 s o es we e comple ely illed. Fig. 4 c shows a simila
app oach bu made in cells p eloaded wi h he Ca21 chela-
o 1,2-bis(2-aminophenoxy)e hane-N,N,N9,N9- e aace ic
acid (BAPTA) o slow he e illing. We can obse e again
ha he e ec o 5 mM ca eine was s ic ly dependen on
he [Ca21]ER a he momen o addi ion. In addi ion, his
expe imen also shows ha Ca21 elease induced by ca -
eine equi es only es ing [Ca21]c. Fu a-2 measu emen s
pe o med in pa allel showed ha in cells loaded wi h
BAPTA, he [Ca21]c changes induced by ca eine we e al-
mos abolished (da a no shown). This esul poin s ou
also ha quan al Ca21 elease by ca eine is due o he eg-
ula ion o Ca21 elease by he lumenal [Ca21], and sug-
ges s ha changes in [Ca21]c do no play a majo ole in he
de elopmen o he quan al e ec .
Ca2
1
En y Ac i a es CICR
The nex s ep in his s udy was o in es iga e he p esence
o CICR ac i a ed by Ca21 en y h ough he plasma
memb ane Ca21 channels. Depola iza ion wi h high K1
medium o by s imula ion wi h nico inic ace ylcholine ago-
nis s such as 1,1-dime hyl-4-phenyl-pipe azinium iodide
(DMPP), p oduced la ge [Ca21]c peaks (Núñez e al.,
1995), ha we e no signi ican ly modi ied by p e ious
Ca21 deple ion o he ER wi h ca eine o wi h he ER
Ca21 pump inhibi o hapsiga gin (see below). The e o e,
he possible con ibu ion o CICR o hese [Ca21]c peaks
canno be es ima ed om con en ional [Ca21]c s udies,
and di ec measu emen o [Ca21]ER becomes essen ial.
Fig. 5 a shows ha 10-s pulses o depola iza ion wi h high
K1 medium induced a ansien Ca21 elease om he
ER, which could be igge ed epe i i ely by consecu i e
pulses. The [Ca21]ER dec ease was o 60–100 mM (10–15%
o he s eady-s a e [Ca21]ER). The e o e, in spi e o he
la ge inc ease in [Ca21]c, he ac i a ion o CICR p oduced
a much smalle [Ca21]ER dec ease han ea men wi h ca -
Figu e 3. Quan al esponse o submaxi-
mal concen a ions o ca eine. The ER
was e illed by pe using wi h medium
con aining 1 mM Ca21 ei he in he p es-
ence (b) o in he absence o ca eine.
Then, di e en concen a ions o ca eine
we e added as indica ed, ei he in he
p esence o in he absence o 10 mM ya-
nodine. O he de ails a e as in Fig. 1.
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The Jou nal o Cell Biology, Volume 144, 1999 246
eine. Inc easing he du a ion o he high K1 pulse in-
c eased he magni ude o Ca21 elease li le. Fig. 5 b illus-
a es he e ec o depola iza ion wi h longe (1 min) high
K1 pulses. In his case, he i s high K1 pulse p oduced
he same e ec as in Fig. 5 a, and hen [Ca21]ER inc eased
mo e apidly, p obably as a esul o he p olonged s imu-
la ion o he ER Ca21 pump by he sus ained high [Ca21]c
le els. This led o a new [Ca21]ER s eady-s a e a z800–900
mM. A e ha , subsequen K1 pulses induced somewha
la ge [Ca21]ER dec eases o 150–200 mM, bu co espond-
ing s ill o only 20% o he s eady-s a e [Ca21]ER. To in es-
iga e i inc eased Ca21 pumping could be esponsible o
he incomple e Ca21 elease, he e ec o K1 depola iza-
ion was es ed in he p esence o he ER Ca21 pump in-
hibi o ciclopiazonic acid (CPA). Fig. 5 c shows ha CPA
i sel induces a slow Ca21 elease om he ER, and ha si-
mul aneous addi ion o high K1 medium induced a as
ini ial Ca21 elease o z20% o he [Ca21]ER, ollowed by
a slowe elease a a a e compa able o ha induced by
CPA alone. This sugges s ha CICR induced by a maxi-
mal K1 depola iza ion is able o p oduce only a dec ease
o z20% o he [Ca21]ER, e en in he absence o Ca21
pumping. CICR, howe e , was po en ia ed by simul a-
neous addi ion o a low ca eine concen a ion. Fig. 5 d
shows ha addi ion o 1 mM ca eine p oduced li le e ec
by i sel , bu s ongly po en ia ed he e ec o K1 depola -
iza ion, ha was now able o elease apidly z50% o he
s o ed Ca21. Finally, CICR could also be igge ed in a
mo e physiological way using an agonis o he nico inic
ace ylcholine ecep o . Addi ion o DMPP induced a apid
and pa ial Ca21 elease om he ER, e y simila o ha
shown abo e o K1 depola iza ion, and which was also
po en ia ed by low concen a ions o ca eine (da a no
shown).
The e ec o a low ca eine concen a ion as posi i e
modula o o CICR migh ep oduce he ac ion o a physi-
ological modula o o his phenomenon. Phospho yla ion
by cAMP-dependen p o ein kinase and p oduc ion o he
b-NAD1 me aboli e cyclic ADP ibose (cADPR) ha e
been epo ed o ac as physiological modula o s o RyR
in bo ine ch oma in cells (Mo i a e al., 1997). Howe e ,
incuba ion o 3–5 min wi h he adenyla e cyclase ac i a o
o skolin (20 mM) had nei he any signi ican e ec on he
sensi i i y o ca eine o Ca21 elease no on he magni-
ude o high K1 depola iza ion-induced CICR (da a no
shown). Rega ding cADPR, i has been epo ed ha ace-
ylcholine, high K1 depola iza ion, and o skolin all s imu-
la e i s syn hesis by ADP ibosyl cyclase in bo ine ch o-
ma in cells (Mo i a e al., 1997). The e o e, p oduc ion o
his media o should al eady be s imula ed in ou CICR
expe imen s. Ne e heless, o s udy di ec ly he e ec o
cADPR on Ca21 elease om he ER, we pe o med ex-
pe imen s in pe meabilized cells. Cells we e deple ed o
Ca21 and econs i u ed wi h coelen e azine as usual. Re-
co ding o luminescence was s a ed and he cells we e
pe meabilized by pe usion wi h in acellula -like medium
con aining 20 mM digi onin and 2 mM EGTA o 1 min.
Then, in acellula -like medium con aining 100 nM Ca21
(bu e ed wi h EGTA) and 2 mM ATP-Mg was pe used.
Fig. 6 a shows ha [Ca21]ER inc eased in digi onin-pe me-
abilized cells wi h e y simila kine ics o ha ound in in-
ac cells a e addi ion o 1 mM ex acellula Ca21. The
s eady-s a e [Ca21]ER eached was also simila (compa e wi h
Fig. 1 a). Fig. 6 a also shows ha 2 mM InsP3 and 50 mM
ca eine p oduced a apid and nea comple e elease o
Ca21 om he ER whe eas 5 mM cADPR had no e ec .
Two di e en comme cial sou ces o cADPR we e es ed
wi h he same esul s. In some expe imen s, cADPR was
added in he p esence o 1 mM calmodulin and no e ec
was ound ei he . A possible explana ion o he disc ep-
ancy among ou esul s and hose o Mo i a e al. (1997)
would be ha cADPR eleases Ca21 om a di e en
(non-ER) Ca21 pool. In ac , hese au ho s epo ha
InsP3 eleases Ca21 om a pool sensi i e o 20 nM hapsi-
ga gin, whe eas cADPR and ca eine elease Ca21 om a
pool only sensi i e o .200 nM hapsiga gin. In ou hands,
e illing o he ER was comple ely inhibi ed by ei he 20 nM
o 1 mM hapsiga gin in bo h in ac (see abo e) and pe -
meabilized cells (da a no shown). The e o e, in o de o
make compa ible ou own esul s and hose o Mo i a e al.
(1997), cADPR and ca eine should be able o elease
Ca21 om an addi ional non-ER Ca21 pool in he p es-
ence o 20 nM hapsiga gin. We hen pe o med single-cell
Figu e 4. Regula ion o ca eine-induced Ca21 elease by
[Ca21]ER. The ER was e illed by pe using wi h medium con ain-
ing 1 mM Ca21 and hen 1 mM his amine o di e en concen a-
ions o ca eine we e added, as indica ed. In he expe imen
shown in c, cells we e incuba ed wi h 10 mM BAPTA-AM du ing
aequo in econs i u ion in o de o load he cy osol wi h his
Ca21 chela o . O he de ails a e as in Fig. 1.
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u a-2 imaging expe imen s in in ac cells looking a he e -
ec o ca eine on [Ca21]c in cells p e ea ed wi h 20 nM
hapsiga gin. Fig. 6 b shows ha addi ion o ca eine o
his amine in Ca21- ee medium p oduced no inc ease in
[Ca21]c unde hese condi ions ( he obse ed dec ease is
due o he pe usion o Ca21- ee medium), whe eas K1
depola iza ion s ill p oduced he usual [Ca21]c peak due o
Ca21 en y.
E ec o Ryanodine on CICR
On he basis o he use-sensi i e ac ion o yanodine, illus-
a ed he e o ca eine s imula ion (Figs. 2 and 3), we
should expec also o ind a use-sensi i e inhibi ion o ER
e illing a e epea ed s imula ion wi h high K1 pulses in-
ducing CICR. Unde his a ionale, expe imen s simila o
hose shown in Fig. 5, bu in he p esence o 10 mM yano-
dine, we e pe o med. In hese expe imen s we we e no
able o de ec any signi ican e ec o yanodine on he
[Ca21]ER dec ease induced by ou o i e consecu i e K1
pulses (da a no shown). Howe e , as aequo in consump-
ion limi s he sensi i i y o he measu emen a he end o
hese expe imen s, we also decided o pe o m single-cell
imaging expe imen s looking a he magni ude o he
[Ca21]c peak induced by ca eine a e se e al K1 pulses in
he p esence o yanodine. Fig 7 a shows ha he [Ca21]c
inc ease induced by ca eine was li le modi ied a e i e
consecu i e 30-s K1 pulses gi en in he p esence o yano-
dine (compa e wi h he ini ial h ee ca eine addi ions). I
is in e es ing o no e ha he second ca eine addi ion a -
e he K1 1 yanodine pulses p oduced no [Ca21]c in-
c ease, e en hough ca eine was always added in he ab-
sence o yanodine. In ac , pe usion wi h yanodine o a
sho pe iod (30–60 s) be o e any ca eine addi ion was e -
ec i e in p omo ing he ypical use-dependen inhibi ion
o ER e illing on applica ion o ca eine (wi hou yano-
dine) 30–60 min la e . This means ha , e en hough yano-
dine by i sel does no p oduce any appa en e ec on ER
illing, i emains wi hin he cells a e washing and ac s
la e , on s imula ion o Ca21 elease (see below). Depola -
iza ion can also be p oduced using he nico inic ace ylcho-
line agonis DMPP ins ead o K1. Again he e, consecu i e
pulses o 10 mM DMPP in he p esence o yanodine did
no ha e any e ec on a la e [Ca21]c peak induced by ca -
eine (da a no shown). An al e na i e and pe haps mo e
physiological depola izing maneu e is ield elec ic s imu-
la ion. Fig. 7 b shows he e ec o se e al consecu i e 10-s
pulses a 10 Hz, be o e and a e he addi ion o yano-
dine. Again, we can obse e ha 5 mM ca eine p oduced
he same [Ca21]c peak a e and be o e ield elec ic s imu-
la ion. As in Fig. 7 a, consecu i e addi ions o ca eine p o-
duced inhibi ion long a e washing o yanodine. The lack
o e ec o yanodine in hese expe imen s may be a ib-
u ed o he much smalle ac i a ion o Ca21 elease by
high K1-induced Ca21 en y compa ed wi h ha induced
by ca eine.
An al e na i e possibili y o ac i a e RyR by Ca21
would be o elease Ca21 di ec ly om he ER using an
InsP3-p oducing agonis in o de o p oduce a big inc ease
in [Ca21]c jus besides he RyR. In ac , i has been e-
po ed ha his amine-induced Ca21 elease could be in-
hibi ed pa ially wi h yanodine a e i e consecu i e
s imula ion pulses (S aude man and Mu awsky, 1991). In
ou hands, s imula ion by i e consecu i e pulses o his a-
mine and yanodine had li le e ec on he i s subsequen
[Ca21]c peak ob ained by ca eine s imula ion (Fig. 7 c). His-
amine was always added in he absence o ex acellula
Ca21, as i also ac i a es Ca21 en y (Cheek e al., 1994b).
Figu e 5. Ac i a ion o
CICR by he Ca21 en y elic-
i ed by high K1-induced cell
depola iza ion. The ER was
e illed by pe using me-
dium con aining 1 mM Ca21.
Then, s anda d medium con-
aining 70 mM KCl was pe -
used as indica ed. In c, 10
mM CPA was added as indi-
ca ed wi h o wi hou high
K1 medium. In his panel,
he [Ca21]ER scale has been
no malized as pe cen age o
he maximum [Ca21]ER le el
in o de o acili a e compa -
ison. In he p esence o CPA
and high K1 medium he ini-
ial a e o [Ca21]ER de-
c ease is much highe ha
wi h CPA alone, bu once
[Ca21]ER is below 80% o he
ini ial le el, he a es o e-
lease in bo h cases u n simi-
la and can be nea ly supe -
imposed. In d, 1 mM ca eine
was also added as indica ed.
O he de ails a e as in Fig. 1.
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The Jou nal o Cell Biology, Volume 144, 1999 248
Simila ly, ca eine was added also in Ca21- ee medium o
a oid he Ca21 en y induced by ca eine, simila o ha
epo ed in GH3 pi ui a y cells (Villalobos and Ga cía-
Sancho, 1996). As be o e, he second ca eine s imula ion
a e his amine and yanodine pulses was comple ely abol-
ished.
E ec s o [Ca2
1
]ER on CICR
In spi e o he lack o inhibi ion by yanodine, he po en i-
a ion by ca eine o Ca21 elease induced by K1 depola -
iza ion (as shown in Fig. 5 d) sugges s ha CICR may ake
place h ough he same RyR ac i a ed by ca eine. An im-
po an p ope y o he RyR ac i a ed by ca eine is he
egula ion by lumenal [Ca21] shown in Figs. 3 and 4. In he
case o CICR, da a o Fig. 5 b also sugges ha Ca21 e-
lease may be s onge a highe lumenal [Ca21] since he
i s s imula ion wi h high K1, ca ied ou when he ER
was only hal - illed, was less e icien han he subsequen
ones. To ob ain mo e e idence on his poin , we in es i-
ga ed he e ec s o 10-s pulses o high K1/Ca21-con aining
medium a di e en [Ca21]ER le els (Fig. 8). A he begin-
ning o he expe imen he ER was comple ely deple ed o
Ca21 and he i s wo K1 pulses induced an inc ease in
[Ca21]ER. Since he cells we e kep in EGTA-con aining
medium du ing he in e als be ween he K1/Ca21 pulses,
e illing o he ER ook place only om Ca21 en e ing
in o he cells du ing he pulses. Subsequen K1 pulses
p oduced also [Ca21]ER inc ease, al hough p og essi ely
smalle , eaching inally a s eady-s a e [Ca21]ER a z300 mM,
whe e he K1/Ca21 pulses had almos no e ec . We hen
comple ely e illed he ER wi h Ca21 by pe using he cells
wi h Ca21-con aining medium, hus eaching he usual
Figu e 6. (a) E ec s o cADPR, InsP3, and ca eine on [Ca21]ER
in pe meabilized cells. Cells we e pe meabilized by pe usion
wi h 20 mM digi onin o 1 min as indica ed. Then he ER was e-
illed by pe usion wi h medium con aining 100 nM Ca21 (bu -
e ed wi h EGTA). Finally, ei he 5 mM cADPR, 2 mM InsP3, o
50 mM ca eine we e pe used as indica ed. (b) E ec o p eincu-
ba ion wi h 20 nM hapsiga gin on he [Ca21]c esponses o his a-
mine, ca eine, and high K1 medium. Cells we e loaded wi h
u a-2, p eincuba ed o 20 min wi h 20 nM hapsiga gin, and
hen suspended in s anda d medium con aining 1 mM CaCl2.
Then, ei he 10 mM his amine, 50 mM ca eine, o s anda d me-
dium con aining 1 mM CaCl2 and 70 mM KCl we e pe used as
indica ed. Bo h his amine and ca eine we e pe used in Ca21-
ee medium (con aining 100 mM EGTA) o a oid Ca21 en y.
Pe usion wi h Ca21- ee medium was s a ed 15 s be o e and
con inued o 15 s a e s imula ion wi h his amine o ca eine.
The ace shown co esponds o he a e age o 42 cells p esen in
he mic oscope ield. O he de ails a e as in Fig. 1.
Figu e 7. E ec s o yanodine and ei he depola iza ion wi h high
K1 medium (a), ield elec ic s imula ion (b), o his amine (c) on
he [Ca21]c esponses induced by ca eine. Cells we e loaded wi h
u a-2 and placed unde he mic oscope in s anda d medium con-
aining 1 mM CaCl2. Then, di e en s imuli we e gi en as indi-
ca ed: di e en concen a ions o ca eine (Ca , in mM), medium
con aining 70 mM KCl (K1), 10 mM his amine (His), 10 mM y-
anodine (Ry), o ield elec ic s imula ion (E.S. 10 Hz o 10 s, a -
ows). Ca eine and his amine we e added in Ca21- ee medium
(con aining 100 mM EGTA). Pe usion wi h Ca21 ee medium
was s a ed 15 s be o e and con inued o 15 s a e he s imuli.
T ansi ion om Ca21-con aining o Ca21- ee medium some imes
p oduced a small [Ca21]c peak. The aces shown a e he a e age
o 36 (a), 49 (b), and 61 (c) cells p esen in he mic oscope ield.
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[Ca21]ER s eady-s a e le els a z700 mM. A ha poin , we
es a ed he p o ocol o epea ed s imula ion wi h 10-s
high K1/Ca21 pulses, using EGTA-con aining medium o
he in e als. The pulses p oduced now a apid Ca21 e-
lease ha could be clea ly dis inguished om he slowe
[Ca21]ER dec ease induced by he lack o ex acellula
Ca21. A e h ee K1 pulses, [Ca21]ER was educed again
o z300 mM, and a ha poin he las pulse p oduced li le
e ec . This esul shows clea ly ha Ca21 elease induced
by K1 depola iza ion, simila ly o ha induced by ca eine,
is s ic ly dependen on he [Ca21]ER.
Di e en ial Inhibi ion o CICR by Type-speci ic Ca2
1
Channel Inhibi o s
We ha e es ed he e ec o se e al inhibi o s o Ca21 en y
on bo h e illing o he ER and high K1 depola iza ion-
induced CICR. A combina ion o inhibi o s o all he ol -
age-dependen Ca21 channels including 1 o 2 h o p ein-
cuba ion wi h -cono oxin GVIA (1 mM), -cono oxin
MVIIC (3 mM), and -aga oxin IVA (1 mM), oge he
wi h p eincuba ion and pe usion along he expe imen
wi h 3 mM nisoldipine, had li le e ec on he a e o e ill-
ing o he ER wi h Ca21. Ins ead, e illing was almos com-
ple ely blocked by pe usion o 100 mM Cd21 (da a no
shown). Nex , he e ec s o he inhibi o s o ol age-ga ed
Ca21 channels on CICR we e es ed. Fo hese pu poses,
we s imula ed he cells wi h high K1 medium in he p es-
ence o 1 mM ca eine (as in he expe imen s shown in Fig.
5 d) in o de o po en ia e he mechanism and inc ease he
sensi i i y o he measu emen s. The dec eases in [Ca21]ER
obse ed, no malized as pe cen age o hose ob ained in
he con ols, we e (mean 6 SEM): con ol, 100 6 11 (n 5
6); 3 mM nisoldipine, 103 6 6 (n 5 5); 1 mM -cono oxin
GVIA, 93 6 6 (n 5 6); 3 mM -cono oxin MVIIC, 42 6 6
(n 5 7); 1 mM -aga oxin IVA, 41 6 9 (n 5 5); a combina-
ion o all h ee oxins and nisoldipine, 20 6 6 (n 5 5).
Toxins and nisoldipine we e p eincuba ed wi h he cells
o 1 o 2 h be o e he measu emen s. When he e ec o
nisoldipine was es ed, his inhibi o was also pe used
along he expe imen .
Ryanodine T ea men Modi ies he [Ca2
1
]c Wa e
Induced by Sho Depola iza ions as Visualized by
Con ocal Mic oscopy
Unde physiological condi ions, cell s imula ion is ig-
ge ed by sho depola iza ions las ing a ew milliseconds.
To es ima e he con ibu ion o CICR o he Ca21 an-
sien unde hese condi ions, we ha e compa ed he a e o
di usion o he Ca21 wa e induced by a sho (100 ms) cell
depola iza ion bo h in con ol cells o in cells in which
he Ca21 s o es had been blocked by p e ious ea men
wi h ca eine and yanodine. We combined he whole-cell
pa ch-clamp echnique wi h luo-3–based mic o luo ime-
y using a con ocal mic oscope. Cells we e line-scanned
along 100-ms squa e depola izing pulses om a holding
po en ial o 270 o 110 mV. The eco ded inwa d cu -
en s showed wo ypical componen s: a ini ial ansien
peak (INa) ollowed by a slow inac i a ing phase (ICa) (da a
no shown). The yanodine ea men did no a ec he o-
al s imula ed Ca21 en y, calcula ed as he in eg al o he
las 90 ms o he eco ded inwa d cu en (mean 6 SEM:
con ol cells, 7.15 6 0.42 pC [n
5 34]; yanodine- ea ed
cells, 6.68 6 1.03 pC [n 5 21]). In spi e o his, line scan im-
ages ep esen ing [Ca21]c showed clea di e ences be-
ween con ol and yanodine- ea ed cells. Fig. 9 a shows
he spa io empo al pa e n o [Ca21]c inc ease in con ol
cells, codi ied in pseudocolo . [Ca21]c inc eased i s nea
he plasma memb ane and hen he Ca21 wa e p opaga ed
in acellula ly. Fig. 9 b shows he esul s ob ained in cells
wi h he Ca21 s o es p e iously emp ied by ea men wi h
ca eine and yanodine. In his case, he [Ca21]c inc ease
was smalle and he p opaga ion o he Ca21 wa e de-
layed. Fig. 9, panels c–e de ail he beha io o se e al pa-
ame e s ha quan i y he phenomenon desc ibed abo e
in e ms o peak [Ca21]c ise (Fig. 9 c), maximum a e o
[Ca21]c inc ease (Fig. 9 d), and ime equi ed o inc ease
luo escence by 10% (Fig. 9 e) a di e en in acellula lo-
ca ions. Fig. 9 c shows ha he maximum luo-3 luo es-
cence (indica ing he maximum [Ca21] peak) was eached
nea he plasma memb ane. An 80% inc ease was ound in
con ol cells compa ed wi h only a 40% inc ease in ya-
nodine- ea ed cells. The luo escence peaks we e smalle
as we mo e deep inside he cell, bu he di e ence among
con ol and yanodine- ea ed cells was main ained. Fig. 9 d
shows ha he maximum a e o luo escence inc ease was
loca ed nea he plasma memb ane and dec eased s eeply
as we mo e in o he cell. Again he e, he a es we e wo o
h ee imes as e in he con ol cells han in he yano-
dine- ea ed ones. Fig. 9 e shows he ime equi ed o he
luo escence o be inc eased by 10% a di e en loca ions.
This pa ame e is e y sensi i e o he in acellula p opa-
ga ion o he [Ca21]c wa e. We ind ha he [Ca21]c wa e
p opaga es abou wice as as in con ol cells han in cells
ea ed wi h yanodine. These esul s indica e ha CICR
signi ican ly con ibu es o he Ca21 signal induced by cell
Figu e 8. E ec o [Ca21]ER on CICR induced by depola iza ion
wi h high K1. Cells deple ed o Ca21 and econs i u ed wi h coe-
len e azine n we e placed in he luminome e in 0.5 mM EGTA
con aining s anda d medium. Then, 10-s pulses o medium con-
aining 70 mM KCl and 2 mM CaCl2 we e gi en as indica ed and
0.5 mM EGTA con aining s anda d medium was pe used du ing
he in e als. A e i e pulses, Ca21-con aining (1 mM) medium
was pe used o 3 min o e ill he ER, and hen he p e ious
p o ocol was s a ed again.
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