Mi ochond ial [Ca
2ⴙ
] Oscilla ions D i en by Local High [Ca
2ⴙ
]
Domains Gene a ed by Spon aneous Elec ic Ac i i y*□
S
Recei ed o publica ion, Augus 16, 2001, and in e ised o m, Sep embe 4, 2001
Published, JBC Pape s in P ess, Sep embe 10, 2001, DOI 10.1074/jbc.C100465200
Ca los Villalobos, Lucı´a Nu´n˜ ez, Pablo Chame o, Ma ı´a Te esa Alonso, and Ja ie Ga cı´a-Sancho‡
F om he Ins i u o de Biologı´a y Gene´ ica Molecula (IBGM), Uni e sidad de Valladolid and Consejo Supe io de
In es igaciones Cien ı´ icas, Depa amen o de Fisiologı´a y Bioquı´mica, Facul ad de Medicina, E-47005 Valladolid, Spain
Mi ochond ia ake up calcium du ing cell ac i a ion
hus shaping Ca
2ⴙ
signaling and exocy osis. In u n,
Ca
2ⴙ
up ake by mi ochond ia inc eases espi a ion and
ATP syn hesis. Ta ge ed aequo ins a e excellen Ca
2ⴙ
p obes o subcellula analysis, bu single-cell imaging
has p o en di icul . He e we combine i us-based ex-
p ession o a ge ed aequo ins wi h pho on-coun ing
imaging o esol e dynamics o he cy osolic, mi ochon-
d ial, and nuclea Ca
2ⴙ
signals a he single-cell le el in
an e io pi ui a y cells. These cells exhibi spon aneous
elec ic ac i i y and cy osolic Ca
2ⴙ
oscilla ions ha a e
esponsible o basal sec e ion o pi ui a y ho mones
and a e modula ed by hypophysio ophic ac o s. Ae-
quo in epo ed spon aneous [Ca
2ⴙ
] oscilla ions in all
he h ee compa men s, bulk cy osol, nucleus, and mi-
ochond ia. In e es ingly, a ac ion o mi ochond ia un-
de wen much la ge [Ca
2ⴙ
] oscilla ions, which we e
d i en by local high [Ca
2ⴙ
] domains gene a ed by he
spon aneous elec ic ac i i y. These oscilla ions we e
la ge enough o s imula e espi a ion, p o iding he ba-
sis o local une-up o mi ochond ial unc ion by he
Ca
2ⴙ
signal.
d ial NADH (12–14) and he syn hesis o ATP (15, 16) and
o he mi ochond ial ac o s equi ed o s imulus-sec e ion
coupling (17).
Aequo in, a Ca
2⫹
-sensi i e pho op o ein, can be di ec ed o a
de ined cellula loca ion by adding speci ic a ge ing sequences,
bu single-cell imaging o aequo in bioluminescence is di icul
because o he e y low ligh ou pu (18). He e we combine
supe b selec i i y o a ge ed aequo in, high exp ession in-
duced by a i al ec o (4, 19), and high sensi i i y p o ided by
a pho on-coun ing came a (20, 21) o esol e changes o [Ca
2⫹
]
in di e en subcellula compa men s a he single-cell le el.
An e io pi ui a y (AP) cells and lines de i ed om hem
(e.g. GH
3
cells) exhibi spon aneous elec ic ac i i y, Ca
2⫹
ac-
ion po en ials, and oscilla ions o he cy osolic [Ca
2⫹
] ([Ca
2⫹
]
c
)
ha a e esponsible o basal AP ho mone sec e ion. Hypophy-
sio ophic ac o s egula e sec e ion by inc easing o dec eas-
ing his spon aneous ac i i y (22, 23). A simila model may
apply o o he exci able sec e o y cells. He e we ha e moni-
o ed [Ca
2⫹
] in di e en subcellula compa men s o li ing AP
and GH
3
cells by bioluminescence imaging o a ge ed aequo -
ins wi h he aim o de ining hei ole in he spon aneous
ac i i y and whe he his may be ele an o he physiologic
unc ion.
EXPERIMENTAL PROCEDURES
Cell Cul u e, [Ca
2⫹
]
c
Measu emen s, and Exp ession o Aequo ins—
Cul u e o GH
3
pi ui a y cells and a AP cells and imaging o [Ca
2⫹
]
c
wi h u a-2 we e as desc ibed p e iously (24, 25). Fo calcula ion o
oscilla ion indexes all he di e ences (in absolu e alue) be ween each
alue and he ollowing one we e added and di ided by he o al numbe
o measu emen s du ing he in eg a ion pe iod. This pa ame e is sen-
si i e o bo h he ampli ude and he equency o oscilla ions (24).
Mi ochond ial aequo in (mi AEQ) and low Ca
2⫹
a ini y mu a ed mi o-
chond ial aequo in ha e been desc ibed p e iously (4). Nuclea and
cy osolic aequo in cDNAs we e ob ained om Molecula P obes and
cloned in he pHSVpUC plasmid. Packaging and i a ion o he pHS-
VnucAEQ (nuclea ) and pHSVcy AEQ (cy osolic) i uses we e pe -
o med as epo ed (19). Cells (3 ⫻10
3
/0.5 ml) we e in ec ed wi h 1–3 ⫻
10
3
in ec ious i us pa icles o a de ec i e he pes simplex i us, ype 1
con aining he co esponding aequo in gene and cul u ed o 12–24 h
be o e measu emen s. In ec ion e iciency anged be ween 22 and 60%
(mean ⫾S.E., 38 ⫾4%; 734 cells om 10 expe imen s).
Imaging o Aequo in Bioluminescence and NAD(P)H Fluo escence—
Cells exp essing apoaequo ins we e incuba ed o 1–2 h a oom em-
pe a u e wi h 1
␮
Mcoelen e azine. Coelen e azine n was used o
econs i u ion o low Ca
2⫹
a ini y mu a ed mi ochond ial aequo in o
dec ease u he he Ca
2⫹
a ini y (26). The s anda d incuba ion me-
dium had he ollowing composi ion (in mM): NaCl, 145; KCl, 5; CaCl
2
,
1; MgCl
2
, 1; glucose, 10; Na-HEPES, pH 7.4, 10. Cells we e placed in o
a pe usion chambe he mos a ized o 37 °C unde a Zeiss Axio e 100
TV mic oscope and pe used a 5–10 ml/min wi h he es solu ions,
p ewa med a 37 °C. A he end o each expe imen cells we e pe me-
abilized wi h 0.1 mMdigi onin in 10 mMCaCl
2
o elease all he esidual
aequo in coun s. Bioluminescence images (20, 21) we e aken wi h a
Hamama su VIM pho on-coun ing came a handled wi h an A gus-20
image p ocesso and in eg a ed o 10-s pe iods. Pho ons/cells in each
image we e quan i ied using Hamama su Aquacosmos so wa e. To al
* This wo k was suppo ed by he Spanish Di eccio´n Gene al de
Ensen˜anza Supe io (DGES; G an s PB97-0474, APC1999-011, and
1FD97-1725-C02-02). C. Villalobos and L. Nu´n˜ ez hold pos doc o al el-
lowships om he Spanish DGES, and P. Chame o holds a p edoc o al
ellowship om he Basque Go e nmen . The cos s o publica ion o his
a icle we e de ayed in pa by he paymen o page cha ges. This
a icle mus he e o e be he eby ma ked “ad e isemen ” in acco dance
wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac .
□SThe on-line e sion o his a icle (a ailable a h p://www.
jbc.o g) con ains Mo ie 1 and Mo ie 2.
‡ To whom co espondence should be add essed: IBGM, Dep . Fisio-
logı´a, Facul ad de Medicina, E-47005 Valladolid, Spain. Tel.: 34-983-
423085; Fax: 34-983-423588; E-mail: [email p o ec ed].
1
The abb e ia ions used a e: [Ca
2⫹
]
M
, mi ochond ial [Ca
2⫹
]; [Ca
2⫹
]
c
,
cy osolic [Ca
2⫹
]; [Ca
2⫹
]
N
, nuclea [Ca
2⫹
]; AP, an e io pi ui a y; TRH,
hy o opin- eleasing ho mone; mi AEQ, mi ochond ial aequo in; cps,
coun s pe second.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 43, Issue o Oc obe 26, pp. 40293–40297, 2001
© 2001 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g 40293
Resea ch on Ca
2⫹
signaling is e ol ing om he global (cel-
lula ) o he local (subcellula ) en i onmen , whe e changes o
[Ca
2⫹
] inside o ganelles also play a p ominen ole (1, 2). Mi-
ochond ia ake up Ca
2⫹
du ing cell ac i a ion (3–5) hus shap-
ing Ca
2⫹
signaling and exocy osis (4, 6, 7). In u n, he inc ease
o mi ochond ial [Ca
2⫹
] ([Ca
2⫹
]
M
)
1
ac i a es se e al mi ochon-
d ial dehyd ogenases (8) hus p o iding a coupling mechanism
o adjus mi ochond ial unc ion o he inc eased needs o ac-
i e cells (5). Close junc ions be ween mi ochond ia and he
endoplasmic e iculum (5, 9, 10) o subplasmalemmal high
[Ca
2⫹
] domains (4, 7, 11) ha e been p oposed o couple local
mi ochond ial unc ion o he Ca
2⫹
signal. In se e al cell sys-
ems s imula ion has been epo ed o inc ease he mi ochon-
This is an open access a icle unde he CC BY license.
coun s pe cell anged be ween 2 ⫻10
3
and 2 ⫻10
5
, and noise was
(mean ⫾S.D.) 1 ⫾1 cps pe ypical cell a ea (2000 pixels). Values e e
o he whole cell a ea. Da a we e i s quan i ied as a es o pho olu-
minescence emission/ o al cps emaining a each ime and di ided by
he in eg a ion pe iod (L/L
TOTAL
in s
⫺1
, whe e Lis luminescence in cps).
Emission alues o less han 4 cps we e no used o calcula ions.
Calib a ions in [Ca
2⫹
] we e pe o med using he alues o he cons an
published p e iously (27). A ansmission image was also aken a he
beginning o each expe imen . Oscilla ion indexes we e calcula ed as
desc ibed abo e o u a-2 bu using he L/L
TOTAL
(s
⫺1
) alues. Mi o-
chond ial NAD(P)H luo escence (14) was measu ed using he same se
up as o aequo in wi h exci a ion a 340 ⫾10 nm and emission a
450 ⫾40 nm. The in eg a ion pe iod was 6 s. Fo hese expe imen s 1
mMpy u a e was added o he s anda d medium o keep he cy osolic
NAD in he oxidized s a e.
RESULTS AND DISCUSSION
To in es iga e whe he mi ochond ial [Ca
2⫹
] oscilla ions
also pa icipa e in he spon aneous ac i i y, pi ui a y cells
we e in ec ed wi h a de ec i e he pes simplex i us ec o ha
exp esses mi ochond ia- a ge ed aequo in (4). Pho on-coun ing
imaging o hese cells (see Fig. 1A, and see Mo ie 1 in Supple-
men al Ma e ial) e ealed clea cu spon aneous [Ca
2⫹
]
M
oscil-
la ions in 40–55% o bo h GH
3
and AP cells (Fig. 1, Band C).
Depola iza ion wi h high K
⫹
solu ion e oked a la ge [Ca
2⫹
]
M
inc ease in he same cells (Fig. 1A,panel K). To explo e
whe he [Ca
2⫹
]
M
oscilla ions could be d i en by Ca
2⫹
en y
seconda y o he spon aneous elec ic ac i i y, we a emp ed
supp ession o elec ical ac i i y by hype pola izing he mem-
b ane in low K
⫹
medium and educing Ca
2⫹
en y by emo ing
ex e nal Ca
2⫹
o blocking he plasma memb ane Ca
2⫹
chan-
nels wi h he dihyd opy idine an agonis u nidipine. We
ound ha all o hese p ocedu es abolished mi ochond ial
Ca
2⫹
oscilla ions (Fig. 1, Band C). Fig. 1Dsumma izes he
esul s o se e al expe imen s quan i ied as oscilla ion indexes,
a pa ame e ha is sensi i e o bo h he ampli ude and he
equency o he oscilla ions (see Re . 24, and see “Expe imen-
al P ocedu es”). Emp ying o he in acellula Ca
2⫹
s o es
wi h hapsiga gin did no dec ease he [Ca
2⫹
]
M
oscilla ions
(Fig. 1D). Fluo escence imaging o u a-2 e ealed spon aneous
[Ca
2⫹
]
c
oscilla ions, which we e also diminished by dec easing
FIG.1.Pi ui a y cells exhibi spon aneous oscilla ions o mi ochond ial [Ca
2ⴙ
]. A, luminescence emission o GH
3
cells in ec ed wi h he
mi ochond ial aequo in i us (pHSVmi AEQ). The i s h ee images (s1–s3) we e aken a di e en imes du ing a 15-min incuba ion in con ol
medium. Calib a ion ma k,10
␮
m. Luminescence in ensi y is coded in pseudocolo (1 o 4 pho ons/pixel) and supe imposed o he g ay
ansmission image aken a he beginning o he expe imen . The ou h image (K) was aken du ing a subsequen 10-s s imula ion wi h high K
⫹
solu ion (150 mM; eplacing he same amoun o Na) and coded in pseudocolo (1–40 pho ons/pixel). Pseudocolo scale is shown a igh . The
in eg a ion pe iod was 10 s o all he images. Also a ailable as Mo ie 1 in Supplemen al Ma e ial. B, e ec s o ex acellula Ca
2⫹
emo al (Ca0;
0.1 mMEGTA added) on spon aneous [Ca
2⫹
]
M
oscilla ions o pHSVmi AEQ-in ec ed AP cells. The aces o 6 single cells ha e been supe imposed.
C, e ec s o low K
⫹
(2.5 mM;K2.5) and TRH (2 nM)on[Ca
2⫹
]
M
oscilla ions o GH
3
cells in ec ed wi h pHSVmi AEQ. The aces o 15 cells ha e been
supe imposed. Also a ailable as Mo ie 2 in Supplemen al Ma e ial. Dand E, oscilla ion indexes (see “Expe imen al P ocedu es”)o [Ca
2⫹
]
M
,
measu ed wi h mi AEQ (D) and o [Ca
2⫹
]
c
, measu ed wi h u a-2 (E) in pHSVmi AEQ-in ec ed GH
3
cells. Measu emen s we e pe o med du ing
incuba ion wi h s anda d medium (Con ), low K
⫹
medium (K2.5), Ca
2⫹
- ee medium (Ca0), 1
␮
M u nidipine (Fu ni), o a e s imula ion wi h 2
nMTRH (measu ed om he 3
d
o he 8
h
min a e TRH addi ion). Thapsi, cells p e ea ed wi h 0.5
␮
M hapsiga gin o 10 min. Each alue is
he mean ⫾S.E. o 23–85 indi idual cells ( wo o ou expe imen s). Resul s a e exp essed as pe cen o he con ols measu ed in he same cells.
All he alues excep Thapsi we e signi ican ly di e en om con ol (p⬍0.001 o p⬍0.05, S uden ’s es ). In AP cells in p ima y cul u e he
esul s we e simila . F, compa ison o he oscilla ion indexes (see “Expe imen al P ocedu es”) o he spon aneous oscilla ions epo ed by
mi ochond ial, cy osolic, o nuclea aequo in in GH
3
cells in ec ed wi h co esponding i uses. Oscilla ion indexes we e compu ed om he
L/L
TOTAL
(s
⫺1
) alues. Each alue is he mean ⫾S.E. o 60–272 cells ( h ee o en expe imen s). Cy osol and nucleus alues we e bo h signi ican ly
smalle han he mi ochond ial ones (p⬍0.01).
Spon aneous Oscilla ions o Mi ochond ial Calcium40294
ex acellula [K
⫹
], Ca
2⫹
emo al, and u nidipine, bu e-
mained una ec ed by emp ying he in acellula Ca
2⫹
s o es
(Fig. 1E). The hypo halamic eleasing ac o TRH, which en-
hances he a e o ac ion po en ial i ing (28), inc eased bo h
he [Ca
2⫹
]
c
and he [Ca
2⫹
]
M
oscilla ions o simila ex en s (see
Fig. 1, C–E; he e ec on [Ca
2⫹
]
M
is also shown in Mo ie 2 in
Supplemen al Ma e ial). Taken oge he , hese esul s sugges
ha he [Ca
2⫹
]
M
oscilla ions a e gene a ed by he [Ca
2⫹
]
c
os-
cilla ions seconda y o he elec ic ac i i y. To in es iga e
whe he his pa e n is ollowed by o he subcellula compa -
men s, we imaged cells exp essing ei he he cy osolic o he
nuclea aequo in. Bo h aequo ins epo ed [Ca
2⫹
] oscilla ions
ha we e dec eased by Ca
2⫹
emo al and inc eased by TRH
(no shown), al hough hey we e much smalle han he mi o-
chond ial ones (Fig. 1F). This sugges s he same o igin o all
he oscilla ions, bu mi ochond ia a e unique in hei abili y o
ampli y he Ca
2⫹
signal. This o ganelle can ake up la ge
amoun s o Ca
2⫹
h ough he mi ochond ial Ca
2⫹
unipo e a
mic omola [Ca
2⫹
]
c
concen a ions (5, 8). Isola ed mi ochond ia
can also accumula e e icien ly Ca
2⫹
h ough he so-called
apid up ake mode when exposed o ains o Ca
2⫹
pulses a
concen a ions abo e 0.4
␮
M(5, 29). In li ing cells, he a e o
up ake is ex emely slow a [Ca
2⫹
]
c
concen a ions below 2–4
␮
M(4, 5). The [Ca
2⫹
]
c
peaks epo ed by ei he u a-2 o cy o-
solic aequo in we e, howe e , below 0.5
␮
M. The e o e, he
la ge [Ca
2⫹
] oscilla ions ound in mi ochond ia sugges ha
local domains wi h highe [Ca
2⫹
]
c
a e ansien ly gene a ed
nea by.
Aequo in bu n-up by Ca
2⫹
can be used no only o de ec
bu also o ace he his o y o high [Ca
2⫹
] domains. In
popula ions o ch oma in cells s imula ed wi h b ie (10-s)
high K
⫹
s imuli wo di e en mi ochond ial pools de elop.
One akes up e y la ge amoun s o Ca
2⫹
(⬎10
⫺4
M) whe eas
he o he accumula es a much smalle Ca
2⫹
load (⬍10
⫺5
M).
Aequo in in he i s pool bu ns ou comple ely du ing he
i s high K
⫹
s imulus and becomes blind o he subsequen
s imuli. The di e en beha io o hese wo pools is no
because o di e en in insic p ope ies bu a he because o
di e en spa ial loca ions ela i e o he plasma memb ane
Ca
2⫹
channels, which de e mines ha hey sense local
[Ca
2⫹
]
c
di e ing by a leas one o de o magni ude (4). He e
we ha e explo ed he beha io o pi ui a y cells, now a he
single-cell le el. When cells we e s imula ed wi h epe i i e
high K
⫹
pulses, nuclea aequo in epo ed a simila ligh
FIG.2.Two mi ochond ial aequo in
pools a e e ealed by depola iza ion
wi h high K
ⴙ
.E ec s o epe i i e s im-
ula ion wi h high K
⫹
on [Ca
2⫹
]
N
, [Ca
2⫹
]
M
,
and [Ca
2⫹
]
c
o GH
3
cells a e shown. Cells
we e s imula ed wi h high K
⫹
(150 mM,
eplacing he same amoun o Na
⫹
) solu-
ion du ing 15 s e e y 2 min. Aand B,
images aken du ing he i s and second
s imulus wi h high K
⫹
in cells in ec ed
wi h ei he he nuclea (A) o he mi o-
chond ial (B) aequo in i us; o he de ails
as in Fig. 1A.Cand D, aequo in consump-
ion (uppe ace) and calib a ed signal
(lowe ace) in cells in ec ed wi h ei he
nuclea (C) o mi ochond ial aequo in i-
uses (D); ci cles ep esen he mean o 29
(C) and 28 (D) single cells; ba s ep esen
S.E. Lines illus a e wo single cells wi h
ex eme beha io s. The a e age con-
sump ion o he i s K
⫹
s imulus was
(mean ⫾S.E.) 53 ⫾6% o he mi ochon-
d ial aequo in (10 expe imen s, 306 cells)
and 4.6 ⫾0.3% o he nuclea aequo in (3
expe imen s, 110 cells). E, measu emen s
o [Ca
2⫹
]
c
in pHSVmi AEQ-in ec ed cells
loaded wi h u a-2 (21); mean ⫾S.E. o 36
single cells. F, cells in ec ed wi h he mu-
a ed, low Ca
2⫹
a ini y, mi ochond ial ae-
quo in i us (pHSVmi mu AEQ) and e-
cons i u ed wi h coelen e azine n; open
symbols, c ude da a (mean ⫾S.E. o 20
single cells); closed symbols, same da a
co ec ed o a pool amoun ing 50% o he
o al aequo in coun s.
Spon aneous Oscilla ions o Mi ochond ial Calcium 40295
ou pu o each s imulus (Fig. 2A), bu mi ochond ial ae-
quo in esponded much mo e s ongly o he i s s imulus
(Fig. 2B). In he nucleus each s imulus p oduced a compa a-
ble aequo in consump ion (2–5% in a e age) co esponding
o nuclea [Ca
2⫹
] ([Ca
2⫹
]
N
) peaks o abou 1
␮
M(Fig. 2C).
Cy osolic aequo in o u a-2 also epo ed ep oducible
[Ca
2⫹
]
c
ises on epe i i e s imula ion (Fig. 2E). In mi ochon-
d ia he i s s imulus consumed abou 40% o he o al
aequo in whe eas each o he ollowing ones consumed only
1–2%, and he i s calib a ed Ca
2⫹
peak was d ama ically
highe han he subsequen ones (Fig. 2D). E en hough
he e was conside able quan i a i e a ia ion among cells,
he quali a i e beha io was simila o all he cells (see
ex eme examples o single-cell aces in Fig. 2, Cand D). In
cells pe meabilized wi h digi onin and exposed o Ca
2⫹
bu -
e s all he mi ochond ial aequo in pool beha ed homoge-
neously (no shown). We in e p e he abo e esul s in e ms
o di e en spa ial loca ion o he wo mi ochond ial pools in
he in ac cells. The i s pool (M1), p obably close o he
plasma memb ane, akes up la ge amoun s o Ca
2⫹
ha bu n
up all i s aequo in and ende i blind o subsequen s imuli.
The second mi ochond ial pool (M2) akes up much smalle
amoun s o Ca
2⫹
, and i s aequo in emains sensi i e o e-
pea ed s imula ion. The a e age sizes o M1 and M2 pools
we e 53 and 47%, espec i ely. To con i m ha blinding o
aequo in in he M1 pool was because o comple e bu n-up by
high [Ca
2⫹
], he expe imen s we e epea ed wi h a mu a ed,
low Ca
2⫹
a ini y aequo in, econs i u ed wi h coelen e azine
n, which enables [Ca
2⫹
] measu emen s in he 30–1000
␮
M
ange (4, 26, 27). Now each s imulus p oduced a ligh ou pu
ha , once co ec ed o he M1 pool size, e ealed epe i i e
[Ca
2⫹
]
M
peaks o abou 400
␮
M(Fig. 2F). A his concen a-
ion he wild ype aequo in would be ⬎90% consumed wi hin
1 s (27). As discussed in de ail elsewhe e (4), he [Ca
2⫹
]
sensed by M1 and M2 pools mus di e by a leas one o de
o magni ude o explain he di e ences in he a es o mi o-
chond ial up ake. The e o e, ou esul s indica e ha subcel-
lula domains wi h e y di e en [Ca
2⫹
]
c
a e gene a ed du -
ing depola iza ion o pi ui a y cells wi h high K
⫹
and ha
hey a e sensed by di e en , s a egically loca ed, mi ochon-
d ial pools. A simila o ganiza ion has been p oposed e-
cen ly in panc ea ic acina cells (7).
Because o physical cons ain s, he high [Ca
2⫹
]
c
domains
mus lie e y close o he plasma memb ane Ca
2⫹
channels
(30). Lack o de ec ion by ei he u a-2 o cy osolic aequo in
also sugges s ha he high [Ca
2⫹
]
c
domains occupy a e y
small ac ion o he o al cy osolic olume. Then, how could
Ca
2⫹
in ade such a la ge ac ion (53%) o he mi ochond ial
pool? I has been shown ecen ly ha con inui y o he in a-
mi ochond ial space is much la ge han hough p e iously (9,
31). The e o e Ca
2⫹
en e ing mi ochond ia nea he plasma
memb ane could di use h oughou he ma ix o in ade
deepe mi ochond ia. On he o he hand, e en a andom dis-
ibu ion o mi ochond ia would esul in p e e en ial loca ion
nea he plasma memb ane. Because o sphe ical shape as
much as 50% o he cell olume lies wi hin 1
␮
m om he
plasma memb ane in a 10-
␮
m-diame e cell.
Do high [Ca
2⫹
]
c
domains also build up in uns imula ed cells?
Can such domains accoun o he spon aneous [Ca
2⫹
]
M
oscil-
la ions? To answe hese ques ions we s udied he e ec s o
blinding he M1 pool wi h a b ie high K
⫹
pulse on he subse-
quen oscilla ions. The high K
⫹
pulse g ea ly dec eased he
oscilla ions epo ed by mi ochond ial aequo in bu no hose
epo ed by u a-2 (Fig. 3, A–C). This sugges s ha spon ane-
ous [Ca
2⫹
]
M
oscilla ions a ise om he M1 pool. The inc ease o
[Ca
2⫹
]
M
o mic omola le els ac i a es se e al mi ochond ial
dehyd ogenases (8). Inc eased ac i i y o mi ochond ial dehy-
d ogenases esul s in inc eased NADH le els (12–14). We ind
ha hype pola iza ion wi h low K
⫹
solu ion, which blocks
[Ca
2⫹
]
M
oscilla ions (Fig. 1C), induced a ep oducible dec ease
o NAD(P)H luo escence (Fig. 3C). Remo al o ex e nal Ca
2⫹
had he same e ec (no shown). These esul s sugges ha he
[Ca
2⫹
]
M
oscilla ions a e egula ing mi ochond ial ac i i y e en
in he basal s a e. Depola iza ion wi h high K
⫹
(Fig. 3, Dand
E) o s imula ion wi h TRH (no shown) p oduced a clea in-
c ease o NAD(P)H luo escence.
A pic u e o a highly s uc u ed spa io empo al o ganiza ion
o Ca
2⫹
signals eme ges om he abo e esul s. Mi ochond ia
con ibu e o shaping local Ca
2⫹
domains (4, 7), bu , in u n,
he Ca
2⫹
signal unes up local mi ochond ial unc ion. A mi o-
chond ial subpopula ion close o he plasma memb ane is able
o moni o he ac i i y o Ca
2⫹
channels, and, by aking up
Ca
2⫹
om local high [Ca
2⫹
]
c
domains, o inc ease [Ca
2⫹
]
M
o
le els high enough o ac i a e mi ochond ial unc ion o ma ch
local ene gy needs (5) and pe haps o p o ide o he ac o s
equi ed o he sec e o y p ocess (17). The emaining mi o-
chond ia, he bulk cy osol, and he nucleus also sense [Ca
2⫹
]
c
oscilla ions bu a a smalle ampli ude, pe haps adequa e o
egula ion o o he cellula unc ions.
FIG.3.Spon aneous [Ca
2ⴙ
]
M
oscilla-
ions ake place in he M1 pool and
egula e espi a ion. A, s imula ion
wi h high K
⫹
(15 s) inhibi s subsequen
spon aneous oscilla ions o mi ochond ial
aequo in luminescence. (GH
3
cells in-
ec ed wi h pHSVmi AEQ; aces om
se en cells ha e been supe imposed). B,
he [Ca
2⫹
]
c
oscilla ions a e no inhibi ed
a e s imula ion wi h high K
⫹
(same
cells as in Aloaded wi h u a-2; a ep e-
sen a i e single cell). C, mean ⫾S.E. o
he oscilla ion indexes (see “Expe imen al
P ocedu es”)o 23–72 cells ( wo o h ee
di e en expe imen s). *, p⬍0.001 (S u-
den ’s es ). D, e ec s o low K
⫹
(2.5 mM),
high K
⫹
(150 mM), and sodium cyanide
(CN;1m
M) on mi ochond ial NAD(P)H
luo escence. All alues exp essed as pe -
cen o he ini ial luo escence. Each da a
poin is he mean ⫾S.E. o 61 cells. Block-
ing cy och ome oxidase wi h CN p omo es
maximal NAD(P)H accumula ion. In i e
simila expe imen s, he dec ease o luo-
escence by incuba ion in low K
⫹
medium
was (mean ⫾S.E.) 6 ⫾1%.
Spon aneous Oscilla ions o Mi ochond ial Calcium40296
Acknowledgmen s—We hank D s. J. Al a ez, J. Llopis, V. L. Lew,
and P. McNaugh on o help ul commen s and J. Fe na´ndez o echni-
cal assis ance.
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