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Regulation of the IP3 Receptor by Ca2+ and Ca2+-Binding Proteins
Laboratory of Physiology
KULeuven
Leuven, Belgium
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VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R RYR
ER/SR
SERCA
IP3R
golgi
PMR1
MitochondriaNCX
Uniporter
Others??
Buffers
R
Second messenger
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IP3R
I, II, III
Agonists
IP3 Ca2+
Intraluminal proteins: Chromogranins; Calnexin, Calreticulin
Cytosolic proteins:
Calmodulin; CaBP
IRBIT;
CARP
Cytoskeletal proteins:
Actin; MyosinII
Ankyrin; Tallin; Vinculin
4.1N
Plasma membrane associated:
Homer-mGluR
TRPs; RhoA-TRPC1
G
Kinases and phosphatases:
PKA; Fyn
BANK- PTK
IRAG-PKG
FKBP12-Calcineurin
PP1
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Ca2+ is the primary modulator of its own release by intracellular Ca2+ release channels
Structure of the IP3R
Regulation of the IP3R by Ca2+
Bell-shaped IICR
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Effect of Calmodulin on IP3-induced Ca2+ release
0%
20%
40%
60%
80%
100%
R1
R2
R3
A7r5 HBE
A7r5 cells
70% IP3R1
0
10
20
30
40
50
60
70
0 0.2 0.4 0.6 0.8
[Ca2+] (µM)
Ca2
+ r
elea
se (
% /
2min
)
CaM
Control
HBE cells
90% IP3R3
0
5
10
15
20
25
0 0.2 0.4 0.6 0.8 1 1.2
[Ca2+] (µM)
Ca2
+ r
elea
se (
% /
2min
)
Control
CaM
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Calmodulin and apocalmodulin
Sienaert, I,. Nadif Kasri, N, Vanlingen S., Parys J. B., Callewaert G., Missiaen, L., and De Smedt, H.
Localisation and Characterisation of a calmodulin/ apocalmodulin binding domain in the N-terminal part of th etype 1 inositol1,4,5- trisphosphate receptor
Biochem. J. 365, 269-277, 2002
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Lbs-1
Lbs-1 1-225
1 581
W226 581
NH2
COOH
ER
CYT
Lbs-1:
IP3 binding core (226-604)
CaM
Ca2+CaM
Recombinant ligand-binding
domain of IP3R1 (LBS-1)
1-225
kDa
62 –
49 –
38 –
28 –17 –14 –
6 –
kDa
62 –
49 –
38 –
28 –17 –14 –
6 –
(Adkins et al., 2000)
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0 5 100.0
0.1
0.2
0.3
B/F
Bound (nM)
0.1 1 100
20
40
60
80
100
[3 H]I
P3
bin
din
g (
%)
[CaM1234] (µM)
EC50= 1.7 µM
Calmodulin effect on IP3 binding
CaM inhibits IP3 binding in Ca2+ -independent way
control ca cam ca cam cam1234 ca cam12340
20
40
60
80
100
[3 H]I
P3 b
indi
ng (
%)
Lbs-1His
Lbs-1 1-225His
1 581
W226 581
Ca2+
Control
CaM1234
Ca2+ /C
aM12
34
Ca2+ /C
aMCaM
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Localisation of a Calmodulin-Binding Site
GST-fusion protein pull down of CaM1234
CaM1234
pGST GST-Cyt1 GST-Cyt2
50 M
free Ca2+
1 mM
EGTACaM1234
Cyt1 Cyt2
Lbs-1
Lbs-1 1-225
1 581
W226 581
1 159 309
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1- B
/Bo
A B C D E F CaM-0,2
0,0
0,2
0,4
0,6
0,8
1,0
200 µM free Ca2+
1 mM EGTA
Detailed localisation using peptides
A B C D E F CaM
A
B
C
E
D
F
1 1591-5-10 1-5-10
1-5-8-14 70% IQ (site1) 76% IQ
53% IQ
A B C D E F CaMCa2+ EGTA
Ca2+ independent CaM-binding site in the N-terminal Region
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Calmodulin binding sites on IP3R1
13 18
3125
Endoplasmic reticulum
Cytosol CaM
R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLAR2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGGR3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA
R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGANR2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQGR3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD
Ca2+/CaM
W1577A (Zhang et al, 2001; Nosyreva et al, 2002)
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10007505002500
600
400
200
0
0.5 1 100
Ca2
+i (
nM
)
Control
CaM
IICR is inhibited by CaM and CaM1234
CaM
CaM 1234
Control
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Regulation of Calcium release By
neuronal Calcium Binding Proteins (CaBP)
Regulation of Calcium release by neuronal Calcium Binding Proteins (CaBP)N. Nadif Kasri, H. Llewelyn Roderick, J.B. Parys, L. Missiaen, H. De Smedt and M. BootmanIn preparation
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Adapted from Haeseleer et al., 2000
CaM or other CaM-like Ca2+ sensor proteins ?
Inhibitory
Activatory ?(Yang et al., 2002)
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CaBP
GST GST1-604
GST1-225
GST226-604
CaBP binds to the InsP3R
NH2
COOH
ER
CYT
IP3 binding core (226-604)
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A
B
C
E
D
F
1 159CaM CaM
sCaBP1 A BC D EF sCaBP1 A BC D EF
+ Ca2+ -Ca2+/ EGTA
A)
B)
CaBP binds to a similar region of the InsP3R as CaM
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CaB
P1/1 1/2 1/4 1/51/6 1/8 1/
10
1/3 CaB
P
Ratio of CaBP: peptide B
0 2 4 6 8 100.0
0.5
1.0
Ban
d in
ten
sity
Peptide B: CaBP
+ Calcium
+ EGTA
Binding of CaBP to the InsP3R is calcium independent
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4 µg Lbs-1
10 µM sCaBP1
5 µM Ca2+
Lbs-1 sCaBP1 Ca Ca/sCaBP1
100[3
H]I
P3
Bin
din
g (
% v
s c
on
tro
l) 100%
78 ± 1.9 %68 ± 6.9 %
39 ± 4.7%
75
50
0
25
CaBP inhibits InsP3 binding to the InsP3R
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Control
0 200 400 600 800 1000
1000
200
400
600
800
0
SCaBPLCaBP
0.5µM 1µM 100µMATP
Time (s)
Ca2+
i (n
M)
Both Long and Short CaBPsinhibit InsP3 induced Calcium release
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CaBP inhibits InsP3 induced Calcium release independent of Calcium binding
EF1 EF2 EF3 EF4
CaBP134
1000
800
600
400
200
0 1500500 1000Time (s)
0
Ca
2+
i (n
M)
0.5 1 100µM ATP
Control
100
50
0% r
es
po
ns
ive
cel
ls
0.5 1 100ATP (M)
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CaBP overexpression inhibits InsP3 Ester induced Calcium release:CaBP acts directly on the InsP3R
200
150
100
120010008006004002000
10 M InsP3 ester
0
50
250
SCaBP1Control
time (s)
Ca
2+
i (n
M)
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Summary
CaBP inhibits IICR
CaBP inhibits IP3 binding
CaBP activity is Calcium independent
CaBP binds on the N-terminal CaM-binding site in a Ca2+-independent way
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??
-15
5
25
45
65
85
105
control IP3 AdPhos CaM sCaBP
Bind
ing
vs c
ontro
l (%
)225-604
GS
TC
ontr
ol
IP
3
Ad
Ph
os
CaM
sCaB
P-1
1-225
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Suramin Interacts with the CaM-binding sites on the IP3R1
1-225
EGTA Ca 2+
IP3R1
Inpu
t
Sep
h
CaM
-Sep
h
+ S
ura
min
Sep
h
CaM
-Sep
h
+ S
ura
min
EGTA Ca 2+
Control
10 µM CaM
0.01 0.1 1 10
0
10
20
30
40
50
Ca2+
rele
ase v
s A
23187 (
%)
[3H]IP3 (µM)
Control
100 µM suramin
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MLCK peptide inhibits IICR
High CaM affinity binding properties
0 2 4 6 8 10 1210
20
30
40
50
60
Fra
cti
on
al
Lo
ss (
%/
2 m
in)
Time (min)
1 µM IP3
1 µM IP3/ 5 µM MLCK peptide
1 µM IP3/ 5 µM MLCK controle peptide
IP3
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0.01 0.1 1 10 100
0
10
20
30
40
50
60
70
80
90
Ca2
+re
leas
e vs
A23
187
(%)
[3H]IP3 (µM)
controle 1 µM MLCK pep 10 µM MLCK pep
0.01 0.1 1 1010
20
30
40
50
60
70
Ca2+
rel
ease
vs
A23
187
(%)
[MLCK peptide] (µM)
IC50: 2 µM
Inhibition of IICR by other CaM-binding peptides?
Properties of the inhibition of IICR by MLCK peptide
0 2 4 6 8 10 12
15
20
25
30
35
40
45
50
55
Fra
ctio
nal L
oss
(%/2
min
)
Time (min)
1 µM IP3
1 µM IP3 + 10 µM RyR peptide
RyR pep: 10 nM
IP3
0 2 4 6 8 10 125
10
15
20
25
30
35
40
45
50
Fra
ctio
nal L
oss
(%/ 2
min
)
Time (min)
control 1 µM Trp3C14 10 µM Trp3C14
Trp3C14: 100 nM
IP3
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Summary
Suramin Inhibits IICR by lowering the IC50 for activation by IP3
MLCK peptide inhibits IICR by lowering Vmax
Suramin interacts with the CaM-binding sites on the IP3R
By removing endogenous CaM?By disrupting intermolecular protein-protein interaction?
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Conclusions
Two CaM-binding sites on the IP3R
Ca2+ dependent in the regulatory domainCa2+ independent in the N-terminus
CaM inhibts IICR only in the presence of Ca2+
Ca2+ is the major regulator of the IP3R
CaM can play an important role in intramolecular protein-protein interactions within IP3R, as removing endogenous CaM inhibits IICR
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A new Calmodulin-dependent CICR mode in A7r5 cells
N. Nadif Kasri, I. Sienaert, Jan B. Parys, G. Callewaert, L. Missiaen, A. Jeromin and H. De Smedt
A novel Ca2+-induced Ca2+-release mechanism in A7r5 cells regulated by calmodulin-like proteins
J. Biol. Chem, 2003 in the press
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0 10 200
10
20
30
40
50
60
70
Fra
ctio
nal l
oss
(%/ 2
min
)
Time (min)
Culture medium Saponin 45Ca2+ TG SDS
4545CaCa2+2+-efflux technique on -efflux technique on permeabilized A7r5 cellspermeabilized A7r5 cells
Fractional loss
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Ca2+ -induced Ca2+ Release in A7r5 cells
0 2 4 6 8 10 12 14 16 18 200
10
20
30
40
50
60
70
80
Frac
tiona
l Los
s (%
/ 2 m
in)
Time (min)
1 µM IP3
3 µM Ca2+
A23187
0
20
40
60
80
100
120
RYRuRedXeC2-APBHEPCTR
Fra
ctio
nal
loss
vs
con
trol
(%
)C
a2+ r
elea
se v
s co
ntro
l
CICR is not mediated via IP3R or RyR
Control Heparin 2-APB XeC RuRed Ryanodine
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0.1 1 10
0
5
10
15
20
25
30C
a2+ r
elea
se v
s A
2318
7 (%
/2 m
in)
[Ca2+] µM
Characteristics of the CICR mode
Ca2+ dependence:
EC50 = 700 nM
Hill = 1.9
Mg2+ inhibition: EC50= 0.6 mM
ATP stimulation: EC50= 320 µM
0.1 1 10
0
5
10
15
20
25
30C
a2+ r
elea
se v
s A
2318
7 (%
/2 m
in)
[Ca2+] µM
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0 10 20
0
10
20
30
40
Time (min)
0.1 1 100
20
40
60
80
100
Ca
2+ r
elea
se v
s con
trol
(%)
[CaM] (µM)
Effects of CaM, CaM1 and CaM1234
control
control
CaM
CaM1
CaM1234
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Calmodulin
Calmodulin1234
Long CaBP1
NCS -1/Frequenin
Short CaBP1
NCS -1/FrequeninE120Q
Calmodulin1
CalmodulinCalmodulin
Calmodulin1234
Long CaBP1
NCS -1/Frequenin
Short CaBP1
NCS -1/FrequeninE120Q
Calmodulin1234
Long CaBP1
NCS -1/FrequeninNCS -1/Frequenin
Short CaBP1
NCS -1/FrequeninE120Q
Calmodulin1
0.1 1 100
20
40
60
80
100
Ca2
+ r
elea
se v
s co
ntro
l (%
)
[CaBP1] (µM)0.1 1 10
0
20
40
60
80
100
Ca2+
rel
ease
vs
cont
rol (
%)
[GST-NCS-1] (µM)
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0 100
10
20
30
40
50F
ract
iona
l los
s (%
/2 m
in)
Time (min)
Ca2+ (3 µM)
control
RyR CaM-BS
(peptide aa 3614-3643)
Preincubation with a CaM-binding peptide inhibits CICR
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0 100
10
20
30
40
50F
ract
iona
l los
s (%
/2 m
in)
Time (min)
CaM but not CaM1234 can restore CICR
Preincubation with
RyR CaM-BS
(peptide aa 3614-3643)
Ca2+ (3 µM)
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0 100
10
20
30
40
50F
ract
iona
l los
s (%
/2 m
in)
Time (min)
CaM but not CaM1234 can restore CICR
Preincubation with
RyR CaM-BS
(peptide aa 3614-3643)
Ca2+ (3 µM)
CaMCaM1234
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Summary
Novel CICR mechanism in A7r5 cells
CICR mechanism is regulated by CaM
IC50: 700 nMActivated by ATPInhibited by MgCl2
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2749
2275
224 576
Gα PIP2
IP3
PLC PLC
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Lab. of Physiology K.U.L., Belgium Brabaham Institute, Cambridge, UK
Ilse Sienaert
Humbert De Smedt
Geert Callewaert
Ludwig Missiaen
Jan B. Parys
Karolina Szlufcik
Geert Bultynck
Sarah Vanlingen
Llewelyn Roderick
Martin Bootman
Andreas Jeromin
Baylor College of Medicine, Houston, Texas