T- ype Ca
2⫹
channels in mouse emb yonic s em cells: modula ion du ing cell
cycle and con ibu ion o sel - enewal
José A. Rod íguez-Gómez,* Kons an ín L. Le i sky,* and José López-Ba neo
Ins i u o de Biomedicina de Se illa; Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e sidad de Se illa, and Cen o de
In es igación Biomédica en Red sob e En e medades Neu odegene a i as, Se ille, Spain
Submi ed 29 July 2011; accep ed in inal o m 27 Oc obe 2011
Rod íguez-Gómez JA, Le i sky KL, López-Ba neo J. T- ype
Ca
2⫹
channels in mouse emb yonic s em cells: modula ion du ing
cell cycle and con ibu ion o sel - enewal. Am J Physiol Cell
Physiol 302: C494–C504, 2012. Fi s published No embe 2,
2011; doi:10.1152/ajpcell.00267.2011.—Ion channels pa icipa e in
cell homeos asis and a e in ol ed in he egula ion o p oli e a ion
and di e en ia ion in se e al cell ypes; howe e , hei p esence and
unc ion in emb yonic s em (ES) cells a e poo ly s udied. We ha e
in es iga ed he exis ence o ol age-dependen inwa d cu en s in
mouse ES cells and hei abili y o modula e p oli e a ion and sel -
enewal. Pa ch-clamped ES cells had inac i a ing e odo oxin (TTX)-
sensi i e Na
⫹
cu en s as well as ansien Ca
2⫹
cu en s abolished by
he ex e nal applica ion o Ni
2⫹
. Biophysical and pha macological
da a indica ed ha he Ca
2⫹
cu en is p edominan ly media ed by
T- ype (Ca
3.2) channels. The numbe o cells exp essing T- ype
channels and Ca
3.2 mRNA le els inc eased a he G1/S ansi ion o
he cell cycle. TTX had no e ec on ES cell p oli e a ion. Howe e ,
blockade o T- ype Ca
2⫹
cu en s wi h Ni
2⫹
induced a dec ease in
p oli e a ion and alkaline phospha ase posi i e colonies as well as
educed exp ession o Oc 3/4 and Nanog, all indica i e o loss in
sel - enewal capaci y. Dec eased alkaline phospha ase and Oc 3/4
exp ession we e also obse ed in cells subjec ed o small in e e ing
RNA-induced knockdown o T- ype (Ca
3.2) Ca
2⫹
channels, hus
pa ially ecapi ula ing he pha macological e ec s on sel - enewal.
These esul s indica e ha Ca
3.2 channel exp ession in ES cells is
modula ed along he cell cycle being induced a la e G1 phase. They
also sugges ha hese channels a e in ol ed in he main enance o he
undi e en ia ed s a e o mouse ES cells. We p opose ha Ca
2⫹
en y
media ed by Ca
3.2 channels migh be one o he in acellula signals
ha pa icipa e in he complex ne wo k esponsible o ES cell
sel - enewal.
ol age-dependen inwa d cu en s; p oli e a ion; Ca
3.2 channel ex-
p ession
FOR THE LAST FEW YEARS emb yonic s em (ES) cells ha e
a ac ed he a en ion o nume ous in es iga o s due o hei
use ulness o de elopmen al s udies and because o hei
abili y o di e en ia e in mul iple cell classes wi h po en ial
applicabili y o egene a i e medicine. Howe e , he basic
physiological ea u es o ES cells a e poo ly known. Mouse ES
cells a e de i ed om he inne cell mass o p eimplan a ion
emb yos. In he p esence o se um and leukemia inhibi o y
ac o (LIF), hese cells can sel - enew, meaning ha hey
p oli e a e main aining an undi e en ia ed and plu ipo en
s a e (5, 47). Plu ipo ency is de ined as he capaci y o gene a e
all he di e en ia ed cell ypes ha cons i u e he adul o gan-
ism. I is known ha p opaga ion o ES cells in an undi e -
en ia ed s a e is go e ned by a complex in e play among a
ne wo k o ansc ip ion ac o s, epigene ic p ocesses, and
in acellula messenge s (35). Howe e , comple e elucida ion
o he mechanisms con olling ES cells sel - enewal and plu-
ipo ency has no ye been achie ed.
ES cells a e hough o be nonexci able, al hough hei
elec ophysiological p ope ies ha e no been ho oughly in-
es iga ed. Vol age- and ligand-ga ed ion channels a e known
o exis in ES cells. In his ega d, hype pola iza ion-ac i a ed
inwa d cu en s, ou wa dly ec i ying K
⫹
cu en s, and GABA-
ac i a ed chlo ide cu en s ha e been epo ed in mouse ES
cells (1, 25, 49). Mo eo e , ol age-dependen K
⫹
cu en s can
be also eco ded om human ES and induced-plu ipo en s em
(iPS) cells (20). Howe e , ol age-ga ed Na
⫹
and Ca
2⫹
chan-
nels, al hough essen ial o nume ous cell unc ions, ha e no
been s udied in de ail in ES cells.
Vol age-ga ed Ca
2⫹
channels a e key playe s o egula ed
Ca
2⫹
in lux in cells (3, 7). Cy osolic Ca
2⫹
concen a ion is a
highly e sa ile in acellula a iable ha egula es a b oad
a ie y o cellula p ocesses, including some o ea ly s ages o
de elopmen , p oli e a ion, di e en ia ion, and apop osis (42,
51). Ca
2⫹
ansien s a e known o occu a he awakening o
quiescence, a he G1/S ansi ion, du ing S-phase, and a he
exi om mi osis (2, 44). In mouse ES cells Ca
2⫹
oscilla ions
ha e also been shown peaking a G1/S ansi ion (22). The e
a e wo majo classes o ol age-dependen Ca
2⫹
channels:
high- ol age ac i a ed (HVA) channels, which include he L-,
N-, P/Q- and R-sub ypes, and low- ol age ac i a ed (LVA),
o T- ype, channels (19, 41). In addi ion o hei dis inc
ol age dependence o ac i a ion, T- ype calcium channels
also exhibi unique apid inac i a ion and slow deac i a ion
ime cou se (41). The T- ype channel amily comp ise h ee
sub ypes, namely Ca
3.1, Ca
3.2, and Ca
3.3, which can be
dis inguished, among o he ea u es, by hei sensi i i y o
Ni
2⫹
(21, 26). The implica ion o T- ype Ca
2⫹
channels in
mechanisms ele an o cell p oli e a ion ha e been de-
sc ibed in di e en umo ( o e iew see Re . 40) and
non umo cells such as he single and wo-cell mouse
emb yo (12), ascula smoo h muscle (24, 43), newbo n
en icula myocy es (17, 30), newbo n ch oma in cells
(29), p eadipocy es (36), and myoblas s (4). In e es ingly, in
a ao ic smoo h muscle cells T- ype Ca
2⫹
cu en s ha e
been shown o change along he cell cycle, because his
ac i i y was p esen only a he G1 and S phases (24).
Al oge he , hese obse a ions p omp ed us o e alua e
whe he ES cells exp ess ol age-ga ed inwa d cu en s and,
mo e speci ically, i T- ype Ca
2⫹
channels pa icipa e in he
physiology o hese highly p oli e a ing cells. Pa icula ly, we
* J. A. Rod íguez-Gómez and K. L. Le i sky con ibu ed equally o his
wo k.
Add ess o ep in eques s and o he co espondence: J. López-Ba neo,
Ins i u o de Biomedicina de Se illa, Campus Hospi al Uni e si a io Vi gen
del Rocío, A da. Manuel Siu o , s/n, 41013 Se ille, Spain (e-mail:
[email p o ec ed]).
Am J Physiol Cell Physiol 302: C494–C504, 2012.
Fi s published No embe 2, 2011; doi:10.1152/ajpcell.00267.2011.
0363-6143/12 Copy igh ©2012 he Ame ican Physiological Socie y h p://www.ajpcell.o gC494
wan ed o know whe he T- ype Ca
2⫹
channels con ibu e o
he main enance o undi e en ia ed s a e o ES cells, o ,
con a ily, induce di e en ia ion. He ein we show ha T- ype
Ca
2⫹
channels a e unc ionally exp essed in mouse ES cells
and ha cells exp essing he Ca
3.2 channel sub ype a e mo e
abundan in he G1/S ansi ion o he cell cycle. We also epo
by bo h pha macological and small in e e ing RNA (siRNA)
ools ha Ca
2⫹
en y media ed by T- ype channels could play
a ole in p ese ing sel - enewal capaci y in mouse ES cells.
We discuss hese esul s in he con ex o he physiological
egula ion o ES cell cycle.
MATERIALS AND METHODS
ES cell cul u e, di e en ia ion, and synch oniza ion. Fou mouse
ES cell lines we e used: AINV15, D3, IB10, and R1. R1 mouse ES
cells we e cul u ed in he p esence o 1,400 U/ml o LIF (Millipo e,
Temecula, CA) and expanded by pe iodical passage in cocul u e wi h
10 ␮g/ml mi omycin C-inac i a ed (Roche Diagnos ics) mouse em-
b yonic ib oblas s o p e en di e en ia ion (27). AINV15 and D3
mouse ES cell lines we e cul u ed and expanded as R1 line wi h mino
modi ica ions in media composi ion (32). IB10 cells we e cul u ed in
medium wi h addi ion o bu alo a li e cells-condi ioned medium.
R1 cells we e used a passages 6, 7, 12, and 13. AINV15, D3, and
IB10 cells we e used a passages 22, 29, and 8, espec i ely. To block
Ca
2⫹
en y and o chela e Ca
2⫹
in cul u e medium, nickel (II) chlo ide
hexahyd a e (NiCl
·
6H
2
O, Sigma-Ald ich) and EGTA (Sigma-Al-
d ich) we e applied espec i ely on ES cell cul u e o 12, 24, o 48 h.
To block Na
⫹
channels e odo oxin (TTX, Sigma-Ald ich) was
applied on ES cell cul u e o 12 h.
Cell-cycle synch oniza ion in G1/S ansi ion was pe o med in all
ES cell lines s udied as p e iously desc ibed (16). B ie ly, cells we e
sequen ially ea ed wi h 2.5 mM hymidine (Sigma-Ald ich), washed
wi h PBS, and ea ed wi h 0.5 mM mimosine (Sigma-Ald ich). A
his poin cells we e dissocia ed and p ocessed ei he o elec ophys-
iological, cell cycle, and mRNA exp ession analysis. Fo elec ophys-
iological s udy, cells we e epla ed on mimosine-con aining medium
o p ese a ion o G1 blockade. Al e na i ely o mimosine, hy-
d oxyu ea (Sigma-Ald ich) was also used.
Fo expansion, cell-cycle blockade and pha macological as well as
siRNA ea men s cells we e cul u ed a high densi y (40,000–62,000
cells/cm
2
) o minimize di e en ia ion caused by low cell densi y. Cell
densi y was main ained cons an o each ype o expe imen .
Elec ophysiology. Mac oscopic Ca
2⫹
and Na
⫹
cu en s we e e-
co ded using he whole cell con igu a ion o he pa ch-clamp ech-
nique as adap ed o ou labo a o y (13, 29). ES cell cul u es we e
dissocia ed wi h ypsin o single-cell suspension and epla ed in
sli e s on poly-L-lysine-coa ed co e slips o elec ophysiological
eco ding 1 o 4 h la e . In hese condi ions mouse emb yonic
ib oblas s, p esen in a much smalle numbe han ES cells, we e
clea ly dis inguishable by hei la ge size. All expe imen s we e
conduc ed a oom empe a u e (22–25°C). Fo whole cell pa ch
eco dings, he in e nal solu ion con ained (in mM) 110 CsCl, 30 CsF,
10 EGTA, 10 HEPES, and 4 ATP-Mg; pH was adjus ed wi h CsOH
o 7.2 and osmolali y was 285 mosM/kg. The s anda d ba h solu ion
con ained (in mM) 140 NaCl, 9 BaCl
2
, 1 CaCl
2
, 10 HEPES, and 10
glucose. pH was adjus ed wi h NaOH o 7.4 and osmolali y was 300
mosM/kg.
Cell cycle analysis. Fo analysis on DNA con en , cells we e ixed
by 70% e hanol d opwise addi ion, ehyd a ed in PBS, and s ained o
p opidium iodide wi h CycleTes PLUS DNA Reagen ki (Bec on
Dickinson, San Jose, CA). Fluo escence in ensi y was analyzed on a
Cy omics low cy ome e (Beckman Coul e , B ea, CA). Da a acqui-
si ion was pe o med using MXP so wa e (Beckman Coul e ), and
he cell cycle pa e n was analyzed wi h FlowJo so wa e (T ee S a ,
Ashland, OR).
Immuno luo escence. Fo immunocy ochemis y, cells we e ixed
wi h 4% pa a o maldehyde/PBS o 20 min, washed in PBS, and hen
blocked o 1ha oom empe a u e wi h 10% no mal donkey se-
um (NDS, Jackson ImmunoResea ch Labo a o ies) and 1% o BSA
(Sigma-Ald ich) in PBS. Nex , cells we e incuba ed wi h monoclonal
mouse an ibody agains s age-speci ic emb yonic an igen 1 (SSEA-1,
1/300, De elopmen al S udies Hyb idoma Bank, Uni e si y o Iowa)
dilu ed in he blocking solu ion o e nigh a 4°C. Cells we e washed
he ollowing day wi h 0.1% BSA-PBS, ea ed wi h pe meabiliza ion
solu ion con aining 0.1% T i on-10% NDS-1% BSA-PBS o 1ha
oom empe a u e, and incuba ed wi h abbi polyclonal Ca
3.2 an i-
body (1/50, gi om D . Fa ach-Ca son) dilu ed in he pe meabili-
za ion solu ion o e nigh a 4°C. Nex , cells we e washed wi h 0.1%
BSA-PBS, incuba ed wi h FITC donkey an i-mouse IgM (Jackson
ImmunoResea ch Labo a o ies) and Alexa 568 donkey an i- abbi IgG
(Molecula P obes, Eugene, OR) seconda y an ibodies, bo h a 1/400 o
1 h a oom empe a u e and washed. To isualize nuclei, cells we e also
s ained wi h 4=-6-diamidino-2-phenylindole (DAPI, 1/1,000, Sigma-
Ald ich). Fluo escence was de ec ed wi h a con ocal mic oscope (TCS
SP2, Leica, Ge many).
Fo low cy ome y, cul u ed cells we e dissocia ed, washed in
PBS, and esuspended wi hou ixa ion in s aining solu ion con aining
( o 50 ml): 44 ml o L15 medium, 100 U/ml penicillin-100 mg/ml
s ep omycin, 10 mM HEPES (all om GIBCO), 0.1 g BSA, and 2.5
mM EDTA (Sigma-Ald ich). Nex , cells we e incuba ed wi h SSEA-1
an ibody (1/300) o 30 min a 4°C and washed and incuba ed wi h
FITC donkey an i-mouse IgM (1/400) o 30 min a 4° C. A e inal
wash, cells we e esuspended in 2 mg/ml 7-amino-ac inomycin (7-
AAD, Molecula P obes) o elimina e dead cells om analyses as
7-AAD⫹. Analyses we e pe o med in a MoFlo h ee lase low-
cy ome e (DAKO Cy oma ion, Fo Collins, CO).
Alkaline phospha ase assay. Cells we e ixed in 4% pa a o malde-
hyde-PBS o 1–2 min, and s aining o alkaline phospha ase was
pe o med a oom empe a u e using an Alkaline Phospha ase De-
ec ion Ki (Millipo e) con aining Naph ol/Fas Red Viole Solu ion as
ecommended by he manu ac u e . Colonies we e obse ed unde an
in e ed mic oscope (IX-71, Olympus, Japan), and he a ios o
undi e en ia ed e sus o al numbe o colonies we e sco ed. Undi -
e en ia ed colonies we e conside ed as hose wi h in ense alkaline
phospha ase s aining and de ined and igh bounda ies.
Gene-exp ession analysis. To al RNA was isola ed om ES cells
using Nucleospin RNA II (Mache ey-Nagel) wi h addi ional RNA p e-
cipi a ion. RNA samples (2–5 ␮g) we e ea ed wi h DNase I (In i ogen,
Ca lsbad, CA) and used o p epa e i s -s and cDNA using Supe Sc ip
II (In i ogen). cDNA samples we e subjec ed o con en ional and
eal- ime PCR ampli ica ion wi h speci ic p ime s. Real- ime PCR
was ca ied ou using an ABI P ism 7500 Sequence De ec ion Sys em
(Applied-Biosys ems, Fos e Ci y, CA) and SYBR G een PCR Mas e
mix (Applied-Biosys ems). In each sample 18S RNA le els we e mea-
su ed o no malize o RNA amoun s and o pe o m ela i e quan i ica-
ions. Fo bo h PCRs, analyses p ime s we e designed using he P ime
Exp ess so wa e (Applied-Biosys ems). Fo con en ional PCR he ol-
lowing p ime s we e used o ampli ica ion o a ge mRNAs: Cacna1g
(Ca
3.1), o wa d 5=-TGGTGGTGGAGAACTTCCATA-3=and e e se
5=-GAAGCAATTACATCGTCCAACA-3=; Cacna1h (Ca
3.2), o wa d
5=-CCGCTATCTGTCCAACGAC-3=and e e se 5=-TACCTTCT-
TACGCCAACGG-3=; Cacna1i (Ca
3.3), o wa d 5=-GTGCCTCGTT-
GTCATAGCGAC-3=and e e se 5=-TGGCCTTGCGAAGGATGT-3=.
Fo eal- ime PCR he ollowing p ime s we e used o ampli ica ion o
a ge mRNAs: Cacna1g, o wa d 5=-TGCTGTGGAAATGGTG-
GTGA-3=and e e se 5=-AGCATCCCAGCAATGACGAT-3=;
Cacna1h, o wa d 5=-TGAGCATGGGTGTTGAGTATCAT-3=and e-
e se 5=-GGTGAACACGATGTTGCTTATCTC-3=; Cacna1i, o wa d
5=-GTGAACATCATGTATAATGGATTGGA-3=and e e se 5=-
TGTGGTTTGTCACCGGCTG-3=; Pou5 1 (Oc 3/4), o wa d 5=-
TCACATCGCCAATCAGCTTG-3=and e e se 5=-GAACCATACTC-
GAACCACATCCTT-3=; Nanog, o wa d 5=-ACCAGTGGTTGAA-
C495T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g
GACTAGCAATG-3=and e e se 5=-CTGGTGCTGAGCCCTTCTG-
3=; 18s, o wa d 5=-AACGAGACTCTGGCATGCTAACTA-3=and
e e se 5=-GCCACTTGTCCCTCTAAGAAGT-3=.
RNA in e e ence. T ansien knockdowns o Ca
3.1 and Ca
3.2
channels we e pe o med wi h siRNAs ob ained om Ambion (Aus-
in, TX). The a ge mRNA sequences o he siRNAs we e he
ollowing: Cacna1g, sense GGAAGAUCGUAGAUAGCAA and
an isense UUGCUAUCUACGAUCUUCCgg; Cacn1h, sense GGGC-
UUCCUUUAGUAGCAA and an isense UUGCUACUAAAGG-
AAGCCCag; sequence o sc amble nega i e con ol siRNA was no
p o ided. Op imiza ion o ES cells ans ec ion condi ions was ca ied
ou by cells ans ec ion wi h a cy omegalo i us-g een luo escen
p o ein (GFP)-exp essing plasmid and subsequen low cy ome y
analysis o GFP-posi i e cells. The bes esul s we e ob ained by cells
ans ec ion 6 h pos pla ing a high cell densi y (62,000 cells/cm
2
).
GFP exp ession le el eached a maximum a 2 days pos ans ec ion
(57% o GFP⫹cells). ES cells we e ans ec ed wi h 20 o 40 nM o
he siRNA duplex using RNAimax lipo ec amine (In i ogen) wi h
p e ious condi ions. siRNA-lipo ec amine complexes we e p epa ed
in Op i-MEM I educed-se um medium (GIBCO, Gai he sbu g, MD).
Cells we e ea ed wi h siRNA-lipo ec amine complexes, and 5 h la e
complexes we e dilu ed wice wi h an ibio ic- ee medium and cul-
u ed o 2 days be o e RNA in e e ence e ec s we e e alua ed.
Knockdown esul s we e ob ained compa ing Ca
3.1 and Ca
3.2
siRNA ea men wi h he co esponding dose o sc amble nega i e
con ol.
Es ima ion o Ca
2
⫹
-bu e ing capaci y o he ES cell cul u e
medium. To e alua e he di alen ca ion bu e ing capaci y o he ES
cells cul u e medium, which could p o ide some e idence on he
chela ion o Ni
2⫹
by he same medium, we used a calcium elec ode
(O ion Calcium Elec ode, The mo). We added 1 mM Ca
2⫹
o he
s anda d solu ion p o ided by he manu ac u e , which had an osmo-
lali y o 150 mosmol/kg. In hese condi ions he esponse o he
elec ode was lineal o added Ca
2⫹
in he ange be ween 0 and 300
␮M. The ES cell cul u e medium, dilu ed o one-hal o each an
osmolali y simila o ha o he s anda d solu ion, had an es ima ed
ee Ca
2⫹
concen a ion ⬃1 mM. In hese condi ions he elec ode
was un esponsi e o added calcium be ween 0 and 75 ␮M and only
s a ed o measu e ee added calcium wi h a lineal cu e simila o he
one obse ed wi h he s anda d solu ion, a e adding Ca
2⫹
a con-
cen a ions highe han 100 ␮M. These measu emen s indica e ha
e en hal -dilu ed ES cell cul u e medium, con aining e al bo ine
se um and o he agen s, has a clea ly mani es ed capaci y o chela e
Ca
2⫹
and possibly o he di alen ca ions.
S a is ical analysis. Values a e gi en as means ⫾SE. The s a is-
ical signi icance o di e ences was analyzed using unpai ed S u-
den ’s - es wi h SigmaS a so wa e.
RESULTS
Inwa d Na
⫹
and Ca
2
⫹
cu en s in mouse ES cells. Mos o
ou expe imen s we e pe o med on R1 mouse ES cells. This
p epa a ion was composed mainly o undi e en ia ed cells
since ⬃90% o hem exp essed SSEA-1 as analyzed by low
cy ome y (Fig. 1A). We ocused on he cha ac e iza ion o
inwa d ol age-dependen ionic cu en s in ES cells. To his
end, ES cells we e dialyzed wi h Cs
⫹
as an ion o subs i u ion
o K
⫹
(13, 29), which abolishes ou wa d ol age-ga ed K
⫹
cu en s desc ibed in hese cells (13, 29).
We ha e dis inguished ou subpopula ions o R1 ES cells
subjec ed o whole cell pa ch clamp (s ep depola iza ion o
⫹20 mV ollowed by epola iza ion o ⫺70 mV), acco ding o
he cha ac e is ics o he inwa d cu en s ha hey exhibi ed
Fig. 1. Inwa d cu en s in mouse emb yonic
s em (ES) cells. A: low cy ome y analysis
pe o med on mouse ES cells o s age-spe-
ci ic emb yonic an igen-1 (SSEA-1, black
line). As nega i e con ol cells we e incu-
ba ed in pa allel wi hou he co esponding
p ima y an ibody (g ay line). B: ep esen a-
i e examples o di e en mac oscopic so-
dium (I
Na
) and calcium (I
Ca
) cu en s e-
co ded in mouse ES cells subjec ed o whole
cell pa ch clamp. C: selec i e inhibi ion o
he Ca
2⫹
ail cu en by ex e nal applica ion
o NiCl
2
(50 ␮M) is shown in he op (g ay
ace). Selec i e inhibi ion o he apid Na
⫹
cu en by ex e nal applica ion o e odo-
oxin (TTX, 200 nM) is shown in he bo om
(g ay ace). The cu en ace in B( op)is
he same as in C( op).
C496 T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g
(see Fig. 1B o ep esen a i e examples). We ha e ound cells
ha showed Na
⫹
and Ca
2⫹
cu en s, only Ca
2⫹
cu en s, only
Na
⫹
cu en s, o no app eciable cu en s. The quan i a i e
analysis gi en in Table 1 indica es ha in no mal condi ions
(asynch onous cul u e) many ES cells (⬃65%) had measu able
inwa d cu en s media ed by Ca
2⫹
and/o Na
⫹
ions. In cells
exp essing Na
⫹
and Ca
2⫹
cu en s he calcium ail cu en was
e e sibly abolished by applica ion o he ex e nal solu ion o
50 ␮MNi
2⫹
, which a his concen a ion selec i ely blocks
T- ype (␣
1H
)Ca
2⫹
channels (26) (Fig. 1C, op). No e ha no
e ec was obse ed on he Na
⫹
cu en . In cells exp essing
Na
⫹
channels, he as sodium cu en was e e sibly blocked
by 200 nM TTX, a well-known speci ic Na
⫹
channel blocke
(Fig. 1C,bo om).
The iden i ica ion o Ca
2⫹
cu en s in mouse ES cells as
T- ype was con i med by hei apid inac i a ion, a dis inc
ea u e o LVA agains HVA Ca
2⫹
channels (41). Figu e 2,A
and B, shows ep esen a i e examples o he deac i a ing Ca
2⫹
cu en s eco ded in R1 mouse ES cells upon epola iza ion o
⫺70 mV a e a depola izing pulse o ⫹20 mV las ing ei he 5
ms (sho pulse) o 50 ms (long pulse). The eco dings clea ly
indica e ha he componen o he ail cu en p esen a he end
o 5 ms pulse disappea ed almos comple ely a he end o he
50-ms pulse, sugges ing inac i a ion o he T- ype channels
du ing he main ained depola iza ion. This es was pe o med
in e e y cell exhibi ing Ca
2⫹
cu en . A slow componen in he
deac i a ion, o closing, ail cu en (indica ed by a ow in Fig.
2C,le ) was consis en ly obse ed, u he sugges ing he
p esence o a popula ion o T- ype Ca
2⫹
channels ha a e
known o close abou ⬃10–20 imes mo e slowly han ei he
Na
⫹
o HVA Ca
2⫹
channels (13, 31). To u he cha ac e ize
he calcium ail cu en s e oked upon epola iza ion, we es i-
ma ed he closing ime cons an (␶) by i ing an exponen ial
unc ion o he deac i a ing segmen o he cu en (Fig. 2C,
igh ). The deac i a ing cu en s we e well i ed by a single
exponen ial unc ion wi h a ime cons an ha a ⫺70 mV
a e aged 1.46 ⫾0.11 ms (see Table 1). This deac i a ion ime
cons an is simila o ha epo ed o na i e and ecombinan
T channels o he Ca
3.2 sub ype exp essing he ␣
1H
subuni
(13, 31). T- ype Ca
2⫹
cu en s o simila kine ics we e e-
co ded in R1 ES cells a ea lie passage as well as in o he
mouse ES cell lines (Table 2; see also below). These obse a-
ions sugges ha mouse ES cells posses a signi ican popula-
ion o ol age-dependen Na
⫹
and Ca
2⫹
channels. The Ca
2⫹
cu en shows ypical kine ics o as inac i a ing and slowly
deac i a ing T- ype channels. The da a also indica e ha he
Ca
2⫹
channels p esen in mouse ES cells p edominan ly belong
o he Ca
3.2 sub ype.
Ca
3.2 T- ype Ca
2
⫹
channels a e induced a G1/S ansi ion
in mouse ES cells. Pha macological blockade o he cell cycle
was used o s udy whe he T- ype Ca
2⫹
channel exp ession is
modi ied du ing ES cell cycle and whe he hey a e p e e en-
ially exp essed in cells a G1/S ansi ion (24). We used a
double blocking s a egy consis ing in a es ing cells a G2/M
phase by hymidine applica ion ollowed by mimosine ea -
men o a es hem a la e G1 phase (16, 50). Mimosine has
been desc ibed as a cell cycle blocke a es ing cells jus a he
e y end o G1 phase, since DNA eplica ion occu s wi hin 15
min o eleasing cells om he mimosine block (23). This
manipula ion o he cell cycle ende ed an accumula ion o
cells in G1/S ansi ion obse ed by an inc ease o cells in G1
Table 1. Elec ical pa ame e s and inwa d ionic cu en s in
asynch onous and G1-synch onized R1 mouse ES cells
Pa ame e Asynch onous Cells G1-Synch onized Cells
I
Ca
⫹I
Na
,% 11.1 (n⫽45) 34.3 (n⫽67)
I
Ca
,% 35.6 (n⫽45) 38.8 (n⫽67)
deac
,ms(a ⫺70 mV) 1.46 ⫾0.11 (n⫽24) 1.49 ⫾0.06 (n⫽50)
I
Ca
, pA/pF (a ⫹20 mV) 4.12 ⫾1.19 (n⫽24) 5.49 ⫾0.57 (n⫽50)
I
Na
,% 17.8 (n⫽45) 4.5 (n⫽67)
I
Na
, pA/PF (a ⫹20 mV) 3.70 ⫾0.68 (n⫽13) 4.40 ⫾0.60 (n⫽26)
No channels, % 35.5 (n⫽45) 22.4 (n⫽67)
Cell capaci ance, pF 5.77 ⫾0.17 (n⫽45) 5.94 ⫾0.13 (n⫽67)
Values o elec ical pa ame e s a e means ⫾SE; n, numbe o cells in
pa en heses. I
Ca
⫹I
Na
, pe cen age o cells wi h inwa d Na
⫹
and/o Ca
2⫹
cu en s, espec i ely; pA/pF: cu en densi y;
deac
: ime cons an o deac i-
a ion.
Fig. 2. T- ype Ca
2⫹
channels in mouse ES cells. A: calcium cu en s om
mouse ES cells du ing sho - (black) and long- (g ay) las ing depola izing
pulses. B:Ca
2⫹
ail cu en s a e sho and long pulses a an expanded ime
scale. No e he disappea ance o he ail gene a ed a he end o 50-ms pulses
(g ay ace) due o inac i a ion o he T- ype channels. C:le , example o
calcium cu en s eco ded in mouse ES cells. Righ ,Ca
2⫹
ail cu en s a an
expanded ime base. Fi ing by a single-exponen ial unc ion (black line) is
shown supe imposed on he cu en ace (g ay). No as componen was
obse ed.
Table 2. Compa ison o T- ype calcium cu en s in di e en
mouse ES cell lines
Cell Line I
Ca
,%
deac
,ms(a ⫺70 mV)
R1 (P13) 46.7 (n⫽45) 1.46 ⫾0.11
R1 (P6) 45.4 (n⫽11) 1.39 ⫾0.05
AINV15 47.5 (n⫽19) 1.98 ⫾0.15
D3 27.3 (n⫽11) 1.03 ⫾0.13
IB10 9.3 (n⫽43) 1.59 ⫾0.14
Values a e means ⫾SE; n, numbe o cells in pa en heses.
C497T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g
phase (25% s. 52%) and a dec ease in S phase (58% s. 36%)
when asynch onous and G1-synch onized cul u es we e com-
pa ed (Fig. 3A,le and igh , espec i ely). Changes o Ca
2⫹
channel exp ession along he cell cycle a e shown in Fig. 3B.
In G1-synch onized cul u es he p opo ion o cells wi hou
Ca
2⫹
channels was lowe han in asynch onous cells (27% s.
53%, espec i ely). Co espondingly, he p opo ion o cells
p esen ing Ca
2⫹
cu en s was highe in G1-a es ed condi ion
han in he asynch onous one (73% s. 47%, espec i ely).
Despi e he inc ease in he numbe o G1-a es ed cells ex-
p essing Ca
2⫹
channels, no changes we e obse ed ei he in
cell size (es ima ed by measu ing cell capaci ance) o in cal-
cium cu en densi y. The ime cons an o T- ype cu en
deac i a ion was also unal e ed (see Table 1).
To check whe he he inc ease in numbe o cells wi h Ca
2⫹
channels was a cell cycle- ela ed e en o a he an a i ac ual
phenomenon caused by mimosine ac ing di ec ly on he chan-
nels, we synch onized he cells in G1/S ansi ion using hy-
d oxyu ea, ano he G1-a es ing agen , ins ead o mimosine. In
his condi ion, a simila p o ile o change in p opo ions o
cells wi h and wi hou Ca
2⫹
channels was ob ained (da a no
shown).
As a es o he undi e en ia ed s a e o he cells du ing cell
cycle a es , exp ession o ma ke s o ES cell plu ipo ency,
Oc 3/4, and Nanog was examined by eal- ime PCR on asyn-
ch onous and G1-synch onized cells. Oc 3/4 and Nanog
mRNA le els inc eased, 11 and 14%, espec i ely, in G1-
synch onized cells bu we can hink o his sligh induc ion as
a ansien change in gene exp ession along cell cycle p og es-
sion (da a no shown). Mimosine-induced G1 blockade was
ully e e sible and a e washing ou he d ug he no mal cell
cycle p o ile o asynch onous cells was ees ablished in G1-
a es ed cells a e an 8-h cul u e pe iod (da a no shown), as i
was p e iously obse ed (23).
The molecula na u e o he T- ype channel class exp essed
in ES cells as well as i s egula ion du ing cell cycle we e
u he analyzed by con en ional and eal- ime PCR. In asyn-
ch onous cells we ampli ied mRNA o he h ee T- ype chan-
nels sub ypes (Fig. 3C). Thei ela i e exp ession le els es i-
ma ed on eal- ime PCR da a we e 0.63 ⫾0.05, 0.36 ⫾0.05,
and 0.01 ⫾0.01 (n⫽4) o Ca
3.1, Ca
3.2, and Ca
3.3,
espec i ely. In G1-a es ed cells, Ca
3.2 mRNA le els in-
c eased signi ican ly (⬃1.7- old), bu he exp ession o he
o he wo channel ypes emained unchanged (Fig. 3D). These
esul s sugges ha al hough bo h Ca
3.1 and Ca
3.2 mRNAs
appea o be abundan in mouse ES cells, he Ca
3.2 sub ype is
he p edominan channel con ibu ing o he T- ype Ca
2⫹
cu en . Molecula and biophysical da a indica e ha his
channel class is selec i ely up egula ed a he G1/S ansi ion
o he cell cycle. Localiza ion o Ca
3.2 channel in he plasma
memb ane was obse ed by immunoci ochemis y in SSEA-1-
posi i e R1 ES cells (Fig. 4).
To asce ain whe he he p esence o T- ype Ca
2⫹
channels
is a gene al p ope y o ES cells, we eco ded ionic cu en s
om se e al ES cell lines g owing in asynch onous s a e. A
ep esen a i e example is shown in Fig. 5A. Al hough he
pe cen age o cells p esen ing Ca
2⫹
channels a ied in he
di e en ES lines s udied (AINV15, D3, and IB10), hey all
seemed o ha e elec ophysiologically simila T- ype Ca
2⫹
cu en s (Table 2). The deac i a ion ime cons an cha ac e is-
Fig. 3. T- ype Ca
2⫹
channels a e ac i e a
G1/S ansi ion o he cell cycle. A: cell cycle
analysis o mouse ES cells in asynch onous
condi ion (le ) and a e G1 synch oniza ion
( igh ). B: numbe o cells wi h and wi hou
Ca
2⫹
cu en s in asynch onous (n⫽45) and
G1-a es ed (n⫽67) cells. Bo h cell ypes
we e coun ed in i e independen expe i-
men s o cell cycle synch oniza ion and di-
ided by he numbe o o al cells. C: exp es-
sion o T- ype Ca
2⫹
channels ansc ip s in
ES cells analyzed by con en ional PCR. Mo-
lecula size o he s anda ds is shown on le .
Re e se ansc ip ion con ol was pe o med
on RNA sample wi hou Supe Sc i p II en-
zyme (RT-). D: gene exp ession le els o
T- ype Ca
2⫹
channels in asynch onous and
G1-a es ed cells analyzed by eal- ime PCR.
The g aph ep esen s he mean alues ⫾SE
(n⫽4). #P⬍0.01.
C498 T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g

ic o T- ype Ca
2⫹
channels was app oxima ely he same in all
cell lines. No majo elec ophysiological di e ences we e
ei he obse ed in T- ype Ca
2⫹
cu en s be ween R1 ES cells a
di e en passage (Table 2). We also s udied T- ype Ca
2⫹
channel mRNA exp ession in AINV15, D3, and IB10 cells. As
in R1 cells (see Fig. 3C), he h ee Ca
3 channel ypes we e
de ec ed in hese ES lines (Fig. 5B). Qui e in e es ingly, he
ela i e exp ession le els o he a ious Ca
3 channel ypes
we e almos he same as hose desc ibed abo e o R1 cells.
Rela i e exp ession le els o Ca 3.1, Ca 3.2, and Ca .3.3,
espec i ely, we e 0.62 ⫾0.06, 0.34 ⫾0.05, and 0.04 ⫾0.01,
(n⫽3) o AINV15; 0.60 ⫾0.07, 0.36 ⫾0.07, and 0.04 ⫾
0.01 (n⫽3) o D3; and 0.75 ⫾0.04, 0.22 ⫾0.05, and
0.03 ⫾0.01 (n⫽3) o IB10 cells. Exp ession o T- ype Ca
2⫹
channels a e cell cycle a es a G1/S ansi ion was also
s udied in AINV15, D3, and IB10 cells. Cell cycle blockade
was pe o med using he same p o ocol as o R1 cells (da a no
shown). Analysis o he wo mos abundan channels (Ca
3.1
and Ca
3.2.) indica ed ha , as in R1 cells, synch oniza ion o
he h ee ES lines esul ed in selec i e inc ease in mRNA
exp ession o Ca
3.2 channels (Fig. 5C). These da a sugges
ha T- ype Ca
2⫹
channel exp ession and hei modula ion
along he cell cycle a e in insic p ope ies o mouse ES cell
lines.
Pha macological blockade o T- ype Ca
2
⫹
channels impai s
sel - enewal o mouse ES cells. As we ound ha Ca
3.2
exp ession is modula ed du ing he cell cycle, we sough
whe he inhibi ion o T- ype Ca
2⫹
channels ac i i y would
ha e an e ec on p oli e a ion and main enance o he undi -
e en ia ed s a e o ES cells. Ni
2⫹
has been demons a ed o be
an e ec i e blocke o he e ologously exp essed ecombinan
T- ype Ca
2⫹
channels wi h highe a ini y o Ca
3.2 han o
Ca
3.1 channels (21). The e o e, we s udied R1 ES cells
ea ed wi h inc easing concen a ions o Ni
2⫹
du ing 12 h
since hei cell cycle is es ima ed o las o ⬃10–12 h. Fo
compa ison, we also s udied he e ec o ex acellula Ca
2⫹
chela ion by addi ion o 5 mM EGTA. The e ec on cell
p oli e a ion o Ca
2⫹
channel blockade and Ca
2⫹
chela ion was
e alua ed by cell coun ing a e T ypan blue exclusion. Ni
2⫹
applica ion (300 ␮M) and EGTA addi ion caused signi ican
dec eases in cell numbe (Fig. 6A). To p ecisely e alua e he
p oli e a ion e ec o Ni
2⫹
and EGTA, he cell cycle pa e n
was analyzed by p opidium iodide s aining ollowed by low
cy ome y (Fig. 6B). We obse ed ha Ni
2⫹
a 150 and 300
␮M caused a signi ican dec ease o cells in he S phase,
sugges ing a dec ease in he p oli e a ion a e, and a small, bu
signi ican , inc ease o cells in sub-G1 phase, p obably indic-
a i e o an apop o ic p ocess. No change was ound in he
pe cen age o cells in he G1 phase a he Ni
2⫹
concen a ions
Fig. 4. Immunocy ochemical analysis o mouse ES cells o SSEA-1 and
Ca
3.2 channel exp ession. Scale ba , 10 ␮m.
Fig. 5. T- ype Ca
2⫹
channels a e induced in di e en ES cell lines a G1/S ansi ion o he cell cycle. A: ep esen a i e example o mac oscopic sodium (I
Na
)
and calcium (I
Ca
) cu en s eco ded in AINV15 ES cell line subjec ed o whole cell pa ch clamp. No e he slow ime cou se o he deac i a ion cu en (I
Ca ail
)
ypical o T- ype Ca
2⫹
channels. B: exp ession o T- ype Ca
2⫹
channel ansc ip s in AINV15, D3, and IB10 ES cell lines analyzed by con en ional PCR.
Molecula size o he s anda ds is shown on he le . Re e se ansc ip ion con ol was pe o med on RNA sample wi hou Supe Sc i p II enzyme (RT-). C: gene
exp ession le els o T- ype Ca
2⫹
channels in asynch onous and G1-a es ed AINV15, D3, and IB10 ES cells analyzed by eal- ime PCR. The g aph ep esen s
he mean alues ⫾SE (n⫽3). *P⬍0.05, #P⬍0.01.
C499T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g
used. Meanwhile, EGTA clea ly induced a mo e po en cell
cycle a es and cell dea h p ocess, as we ound a signi ican
dec ease o cells in S phase and inc ease o sub-G1 and G1
ac ions. Di e ence be ween Ni
2⫹
and EGTA e ec s on cell
cycle pa e n unde lines he sub le change on cell p oli e a ion
p o oked by Ca
2⫹
channels blockade compa ed wi h Ca
2⫹
chela ion. The magni ude o changes p o oked by exposu e o
Ni
2⫹
and Ca
2⫹
chela ion on cell p oli e a ion inc eased when
he ea men was expanded om 12 h o 24 o 48 h, possibly
due o p olonged di e en ia ion and cell dea h (Fig. 6A). Since
we obse ed an inc eased p esence o Na
⫹
channels in G1/S
synch onized cells (see Table 1), we s udied he e ec o Na
⫹
channel blockade (TTX was applied a 200 and 500 nM du ing
12 h) on p oli e a ion. No signi ican changes we e obse ed in
any o he phases o he cell cycle, hus sugges ing ha Na
⫹
channels do no signi ican ly pa icipa e in cell p oli e a ion
(da a no shown).
Sel - enewal o Ni
2⫹
- and EGTA- ea ed ES cells in he
12-h ime poin was e alua ed by alkaline phospha ase ac i i y
assay and eal- ime PCR o plu ipo ency ma ke genes Oc 3/4
and Nanog. Ni
2⫹
applica ion (a 150 and 300 ␮M) caused
signi ican dec eases in he numbe o alkaline phospha ase-
posi i e undi e en ia ed colonies (Fig. 6C). EGTA had a
s onge e ec and led o almos abolishmen o he p esence o
undi e en ia ed colonies. Simila ly, mRNA le els o Oc 3/4
and Nanog we e down egula ed by Ni
2⫹
(a 150 and 300 ␮M),
as well as by EGTA ea men (Fig. 6D). Taken oge he , hese
esul s sugges ha blockade o T- ype Ca
2⫹
channels de-
c eases p oli e a ion and sel - enewal o mouse ES cells.
RNA in e e ence o Ca
3.2 channels induce ES cell di e -
en ia ion. To assess speci ically he ole o T- ype Ca
2⫹
channels
on ES sel - enewal an RNA in e e ence assay was pe o med
wi h siRNA exp ession plasmids o Ca
3.1 and Ca
3.2, he mos
abundan ly exp essed T- ype Ca
2⫹
channels in ES cells. In e e -
ence e iciency was es ed by eal- ime PCR o he a ge gene in
cells ans ec ed wi h sc amble nega i e con ol, Ca
3.1, and
Ca
3.2 siRNAs (Fig. 7A). A e ans ec ion wi h siRNA o
Ca
3.1 a 40 nM, mRNA le el o he a ge gene was educed o
25%, whe eas Ca
3.2 mRNA le els we e nonspeci ically de-
c eased o 75% o con ol alues. Meanwhile, siRNA o Ca
3.2
a 20 and 40 nM educed exp ession le els o he a ge gene o
43% and 40%, espec i ely. Ca
3.2 siRNA ea men also caused
a nonspeci ic dec ease in mRNA le els o Ca
3.1 channels.
Down egula ion o Ca
3.1 and Ca
3.2 mRNA caused no signi i-
Fig. 6. Ni
2⫹
ea men impai s sel - enewal o mouse ES cells. A: quan i ica ion o cells ea ed wi h inc easing doses o Ni
2⫹
andaCa
2⫹
chelan , EGTA (5 mM)
o 12, 24, and 48 h. The g aph ep esen s means ⫾SE (n⫽6). #P⬍0.01. B: cell cycle analysis o ES cells ea ed o 12 h wi h inc easing doses o Ni
2⫹
and EGTA (5 mM). The g aph ep esen s he means ⫾SE (n⫽6). *P⬍0.05, #P⬍0.01. C: analysis o alkaline phospha ase ac i i y on ES cells exposed o
inc easing doses o Ni
2⫹
and EGTA (5 mM) o 12 h. The g aph ep esen s he means ⫾SE (n⫽4). #P⬍0.01. Rep esen a i e ields o cells o con ol (C
1
),
Ni
2⫹
150 ␮M(C
2
), Ni
2⫹
300 ␮M(C
3
), and EGTA 5 mM (C
4
) condi ions and s ained o alkaline phospha ase a e shown (bo om). Scale ba , 50 ␮M.
D: gene exp ession le els o ES cells ma ke s Oc 3/4 and Nanog in ES cells ea ed wi h inc easing doses o Ni
2⫹
and EGTA (5 mM) o 12 h. Analysis was
pe o med by eal- ime PCR. The g aph ep esen s he means ⫾SE (n⫽4). *P⬍0.05, #P⬍0.01.
C500 T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g
can change in cell g ow h o cell cycle pa e n o siRNA- ea ed
cells (da a no shown). Howe e , he pe cen age o alkaline
phospha ase-posi i e undi e en ia ed colonies dec eased signi i-
can ly o cells ans ec ed wi h 40 nM Ca
3.2 siRNA bu no o
40 nM Ca
3.1 siRNA ans ec ion (Fig. 7B). Exp ession le els o
ES cell ma ke s we e educed signi ican ly o Oc 3/4 bu no o
Nanog in cells ans ec ed wi h 20 nM Ca
3.2 siRNA (Fig. 7C).
Sligh dec eases in bo h genes we e ound in cells ea ed wi h 40
nM Ca
3.1 siRNA, bu none o hem eached signi icance. These
esul s suppo he iew ha T- ype cu en a ibu ed o Ca
3.2
channels is pa icipa ing in he mechanism esponsible o main-
enance o mouse ES cells sel - enewal.
DISCUSSION
The elec ophysiological p ope ies o ES cells a e poo ly
de ined, hence, his epo aimed a he cha ac e iza ion o he
inwa d cu en s p esen in hese cells. Memb ane ion channels
play undamen al oles in a my iad o cellula p ocesses,
including p oli e a ion and di e en ia ion. We ha e ound ha
se e al lines o undi e en ia ed mouse ES cells ha e ol age-
dependen Ca
2⫹
and/o Na
⫹
cu en s. Molecula and elec o-
physiological e idence indica e ha he Ca
2⫹
cu en s a e
almos exclusi ely media ed by LVA o T- ype Ca
2⫹
channels.
We show ha T- ype channels a e modula ed along cell cycle
p og ession and con ibu e o main enance o ES cells sel -
enewal. ES cell sel - enewal, which is widely de ined as
p omo ion o p oli e a ion accompanied by supp ession o
di e en ia ion (5, 47), is equi ed in o de ha plu ipo ency is
e ained. De ailed cha ac e iza ion o he signaling pa hways
ope a ing in sel - enewal and plu ipo ency will con ibu e o
exploi he ull po en ial o ES cells.
ES cells a e known o exp ess ou wa d K
⫹
cu en s and
hype pola iza ion-ac i a ed inwa d cu en s (49), bu o ou
knowledge, his is he i s epo whe e unc ional ol age-
ga ed Ca
2⫹
and Na
⫹
channels we e eco ded in hese cells. In
a ecen pape (46), mRNA o a ious Ca
2⫹
channel classes (L,
N, R, and T) we e ampli ied by PCR om p epa a ions o
mouse ES cells; howe e , whe he hese mRNAs a e ansla ed
o unc ional channels was no analyzed. In he cu en s udy,
we ha e shown ha among he a ious classes o HVA o LVA
Ca
2⫹
channels, only T- ype channels a e indeed unc ional in
he mouse ES cell lines s udied. The deac i a ion ime cou se
and sensi i i y o Ni
2⫹
sugges ha o he h ee T- ype mRNA
species ampli ied by PCR, Ca
3.2 channels (␣
1H
subuni ) a e
he main con ibu o s o he Ca
2⫹
cu en in mouse ES cells
(21, 26, 31). We also ound se e al elec ophysiologically
dis inc subpopula ions o undi e en ia ed ES cells depending
on whe he hey exp ess Na
⫹
channels, Ca
2⫹
channels, bo h, o
none o hem. Al hough he unc ional ole o he Na
⫹
cu en s
was no s udied in de ail, Ca
2⫹
channel unc ion seems o be
associa ed wi h main enance o p oli e a ion and undi e en i-
a ed s a e o he cells.
Blockade o T- ype Ca
2⫹
channel wi h Ni
2⫹
(a 150 and 300
␮M) du ing 12 h was su icien o dec ease p oli e a ion a e
and p o oke a subs an ial loss in R1 ES cells sel - enewal,
obse ed as educ ion in he pe cen age o undi e en ia ed
colonies, and dec eased exp ession o plu ipo ency ma ke s
Oc 3/4 and Nanog. Ni
2⫹
applica ion induced mode a e de-
Fig. 7. RNA in e e ence o Ca
3.2 induces
di e en ia ion o mouse ES cells. A: gene
exp ession le els o Ca
3.1 and Ca
3.2 genes
in cells ea ed wi h sc amble nega i e con-
ol (a 20 and 40 nM), Ca
3.1 a 40 nM, and
Ca
3.2 (a 20 and 40 nM) small in e e ing
RNA (siRNAs). Analysis was pe o med by
eal- ime PCR. The g aph ep esen s he
means ⫾SE (n⫽4). *P⬍0.05, #P⬍0.01.
B: analysis o alkaline phospha ase ac i i y in
mouse ES cells ea ed wi h sc amble nega-
i e con ol, Ca
3.1, and Ca
3.2 siRNAs all a
40 nM. The g aph ep esen s he means ⫾SE
(n⫽4). *P⬍0.05. Rep esen a i e ields o
cells ea ed wi h 40 nM sc amble nega i e
con ol (B
1
), Ca
3.1 (B
2
), and Ca
3.2 siRNAs
(B
3
) and s ained o alkaline phospha ase a e
shown (bo om). Scale ba , 50 ␮M. C: gene
exp ession le els o ES cells ma ke s Oc 3/4
and Nanog ea ed wi h sc amble nega i e
con ol (a 20 and 40 nM), Ca
3.1 a 40 nM
and Ca
3.2 siRNAs a 20 nM. Analysis was
pe o med by eal- ime PCR. The g aph ep-
esen s he means alues ⫾SE (n⫽4). #P⬍
0.01.
C501T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g
c ease o cells in S phase, which is an indica ion o ele a ed ES
cell di e en ia ion (45, 52). On he o he hand, inc ease in
sub-G1 ac ions a 150 and 300 ␮MNi
2⫹
could be indica i e
o apop o ic cell dea h, an e en also associa ed o blas ocys
de elopmen and LIF wi hd awal-induced mouse ES cell di -
e en ia ion (10, 15, 33, 39). Al e na i ely, apop osis could be
caused by oxici y induced by Ni
2⫹
exposu e (6). I is impo -
an o no e ha he di e en ia ion e ec induced by Ni
2⫹
was
only clea ly obse ed a concen a ions an o de o magni ude
highe han hose equi ed o selec i ely block ecombinan
Ca
3.2 channels (IC
50
⬃5–10 ␮M) (21). I is he e o e possible
ha na i e Ca
3.2 channels exp essed in ES cells ha e lowe
appa en a ini y o Ni
2⫹
han he he e ologously exp essed
channels. An addi ional ci cums ance ha could explain he
ela i ely high Ni
2⫹
concen a ions equi ed o al e ES cell
sel - enewal is ha ee Ni
2⫹
concen a ion could be less han
he p esumed nominal alue due o di alen ca ion chela ion by
componen s (such as e al bo ine se um) o he ES cell cul u e
medium. Indeed, a powe ul Ca
2⫹
-chela ing capaci y o he
cul u e medium was obse ed (see MATERIALS AND METHODS).
In acco d wi h he pha macological da a, Ca
3.2 siRNA
ea men induced a modes , bu s a is ically signi ican , loss o
sel - enewal obse ed as a dec ease in he pe cen age o
undi e en ia ed colonies and Oc 3/4 mRNA exp ession le el.
Because o he lowe changes in he undi e en ia ed s a e, he
p oli e a ion capaci y o ES cells emained unchanged. The
ela i ely mode a e e ec o Ca
3.2 knockdown on ES sel -
enewal compa ed wi h Ni
2⫹
applica ion can be explained by
he limi ed e iciency o siRNA o inhibi he Ca
3.2 ansc ip
(60% educ ion o mRNA exp ession). I is also no ewo hy
ha in pa allel expe imen s Ca
3.1 knockdown did no cause
any signi ican change on he sel - enewal pa ame e s o ES
cells.
The mos plausible ole o T- ype Ca
2⫹
channels in ES cells
is o egula e ansmemb ane Ca
2⫹
in lux, which could, in u n,
in luence he exp ession o he gene p og am, o signaling
pa hways necessa y o p oli e a ion and sel - enewal. The
pa icula biophysical ea u es o T- ype Ca
2⫹
channels allow
hem o open a memb ane po en ials sligh ly mo e nega i e
han hose a which hey ully inac i a e. In a memb ane
po en ial ange nea he cell es ing po en ial he e is a small
pe cen age o channels ha a e no inac i a ed and can media e
small sus ained inwa d Ca
2⫹
cu en s (“window cu en s”) (9,
11). The e o e, in ES cells T- ype Ca
2⫹
channels could ans-
la e sub le changes in memb ane po en ial in o modi ica ions o
ansmemb ane Ca
2⫹
in lux and inc ease o cy osolic Ca
2⫹
concen a ion. Indeed, i is known ha memb ane po en ial is a
c i ical ac o o cell cycling as blockade o K
⫹
channel
ac i i y leads o memb ane depola iza ion, a es in ea ly G1,
and dec eased p oli e a ion a e in di e en cell ypes, includ-
ing mouse ES cells and human iPS cells (20, 34, 38, 49,
53–55). The ole o in acellula Ca
2⫹
oscilla ions a he G1/S
ansi ion and p oli e a ion has been highligh ed in mouse ES
cells (22). This las s udy epo ed ha ex acellula Ca
2⫹
does
no seem necessa y o ins au a ion o Ca
2⫹
oscilla ions bu o
he main enance o hei ampli ude, since oscilla ion was ig-
ge ed by elease o Ca
2⫹
om endoplasmic e iculum in an
inosi ol 1,4,5- isphospha e-media ed mode. We ha e obse ed
ha Ca
3.2 channels a e up egula ed a he G1/S ansi ion,
he e o e, i is likely ha Ca
2⫹
in lux media ed by T- ype
channels in luence he ampli ude o in acellula Ca
2⫹
oscilla-
ions as cells p og ess om G1 o S phase. This can be
pa icula ly impo an o cell cycle egula ion since i has been
p o en ha he ampli ude and du a ion o Ca
2⫹
signals de e -
mine he ac i a ion o speci ic ansc ip ional pa hways (14).
Mouse ES cells a e cha ac e ized by ex ao dina ily apid
p oli e a ion a e, p ima ily owing o a educ ion in he du a-
ion o G1 phase o he cell cycle. De ining ea u es o G1
phase in ES cells a e cons i u i e cyclinE-CDK2 ac i i y and
hype phospho yla ed e inoblas oma p o ein (37). Inc eased
in acellula Ca
2⫹
le el has been demons a ed o oscilla e a
di e en ime poin s o he cell cycle including he G1/S
ansi ion (2, 44). Di e en signaling pa hways could in ol e
T- ype Ca
2⫹
channels as plausible cell cycle egula o s in ES
cells a G1/S ansi ion. T- ype Ca
2⫹
cu en s ha e been shown
media ing pheno ype swi ching o nonp oli e a i e o p oli e -
a i e smoo h muscle cells, and calmodulin, an in acellula
calcium ecep o , has been shown o pa icipa e in his ansi-
ion (18). Calmodulin has also been demons a ed o acili a e
G1/S ansi ion in ascula smoo h muscle cells ia binding o
cyclinE and possibly ac i a ing CDK2 (8). A di e en pa hway
would in ol e calmodulin as an induce o e inoblas oma
p o ein phospho yla ion (48). Ei he by modula ing cons i u-
i e cyclinE-CDK2 ac i i y o by main aining e inoblas oma
hype phospho yla ion T- ype Ca
2⫹
channels ac i i y could
acili a e he ansi ion om G1 o S phase in ES cells.
In conclusion, we show a no el ea u e o ES cells, which is
he p esence o Na
⫹
and T- ype Ca
2⫹
cu en s, he las ones
media ed by Ca
3.2 channels up egula ed in he G1/S ansi-
ion o he cell cycle. These channels seem o pa icipa e in he
main enance o he undi e en ia ed s a e o mouse ES cells.
Taking in o accoun ha hypoxia induces Ca
3.2 channels in
cul u ed cells (13), i would be in e es ing o es whe he he
e ec desc ibed he e ope a es also in human ES and iPS cells
conside ing ha hypoxia main ains plu ipo ency o human ES
cells and imp o es e iciency o adul cell ep og amming (28,
56). Fu he s udies on he mechanisms linking ion channel
ac i i y, cell cycle egula ion, and ansc ip ional ne wo ks
may imp o e ou unde s anding o ES cells sel - enewal and
plu ipo ency.
ACKNOWLEDGMENTS
Monoclonal Ca
3.2 an ibody was a kind gi o D . Ma y C. Fa ach-Ca son
(Uni . o Delawa e). We hank D . Juan Tejedo (Cabime , Se illa) o kindly
p o iding AINV15 and D3 ES cell lines, D . Ca men Saez (Ins i u o de
Biomedicina de Se illa) o helping us o es ablish cell cycle synch oniza ion
expe imen s, and o T ee S a o kindly p o iding us ee u iliza ion o FlowJo
so wa e.
GRANTS
This esea ch was suppo ed by he Andalusian Go e nmen , Spanish
Minis y o Science and Heal h and Ma celino Bo ín Founda ion.
DISCLOSURES
No con lic s o in e es , inancial o o he wise, a e decla ed by he au ho (s).
AUTHOR CONTRIBUTIONS
Au ho con ibu ions: J.A.R.-G., K.L.L., and J.L.-B. concep ion and design
o esea ch; J.A.R.-G. and K.L.L. pe o med expe imen s; J.A.R.-G., K.L.L.,
and J.L.-B. analyzed da a; J.A.R.-G., K.L.L., and J.L.-B. in e p e ed esul s o
expe imen s; J.A.R.-G. and K.L.L. p epa ed igu es; J.A.R.-G. and J.L.-B.
d a ed manusc ip ; J.A.R.-G., K.L.L., and J.L.-B. edi ed and e ised manu-
sc ip ; J.A.R.-G., K.L.L., and J.L.-B. app o ed inal e sion o manusc ip .
C502 T-TYPE Ca
2⫹
CHANNEL ACTIVITY IN MOUSE EMBRYONIC STEM CELLS
AJP-Cell Physiol •doi:10.1152/ajpcell.00267.2011 •www.ajpcell.o g