1. Regulation of the IP3 Receptor by Ca2+ and Ca2+-Binding Proteins
Laboratory of Physiology
KULeuven
Leuven, Belgium

2. R LGCC VGCC SOCC MSCC NCX IP3R RYR NCX PMCA SERCA Others?? ER/SR PMR1
Second
messenger
IP3R
RYR
NCX
Mitochondria
PMCA
Others??
IP3R
ER/SR
PMR1
SERCA
Uniporter
golgi
Buffers

3. Agonists IP3 Ca2+ IP3R I, II, III Cytoskeletal proteins:
Actin; MyosinII
Ankyrin; Tallin; Vinculin
4.1N
Agonists
IP Ca2+
Plasma membrane associated:
Homer-mGluR
TRPs; RhoA-TRPC1
G
Kinases and phosphatases:
PKA; Fyn
BANK- PTK
IRAG-PKG
FKBP12-Calcineurin
PP1
Cytosolic proteins:
Calmodulin; CaBP
IRBIT;
CARP
IP3R
I, II, III
Intraluminal proteins: Chromogranins; Calnexin, Calreticulin

4. Regulation of the IP3R by Ca2+
Structure of the IP3R
Bell-shaped IICR
Ca2+ is the primary modulator of its own release by intracellular Ca2+ release channels

5. Effect of Calmodulin on IP3-induced Ca2+ release
A7r5 cells
70% IP3R1
CaM
Control
HBE cells
90% IP3R3
Control
CaM
A7r5
HBE

6. 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, , 2002

7. Lbs-1: IP3 binding core (226-604) Lbs-1  1-225
(Adkins et al., 2000)
CaM
NH2
CYT
Ca2+CaM
COOH ER
Recombinant ligand-binding
domain of IP3R1 (LBS-1) 1
581
kDa 62 – 49 38 28 17 14 6
Lbs-1
1-225
W226
581
Lbs-1  1-225

8. Calmodulin effect on IP3 binding
[3H]IP3 binding (%)
Lbs-1His
Lbs-1  1-225His 1
581
W226
Ca2+
Control
CaM1234
Ca2+/CaM1234
Ca2+/CaM
CaM
EC50= 1.7 µM 5 10
0.0
0.1
0.2
0.3
B/F
Bound (nM)
CaM inhibits IP3 binding in Ca2+ -independent way

9. GST-fusion protein pull down
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
EGTA
Cyt Cyt2
Lbs-1
Lbs-1  1-225 1
581
W226
159
309

10. Detailed localisation using peptides
A B C D E F CaM A B C E D F 1
159
1-5-10
70% IQ (site1)
76% IQ
53% IQ
A B C D E F CaM
Ca2+
EGTA
1- B/Bo
Ca2+ independent CaM-binding site in the N-terminal Region

11. Calmodulin binding sites on IP3R1
R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN
R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG
R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD 13 18 31 25
Endoplasmic reticulum
Cytosol
CaM
W1577A (Zhang et al, 2001; Nosyreva et al, 2002)
R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA
R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG
R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA
Ca2+/CaM

12. IICR is inhibited by CaM and CaM1234
Control
0.5 1
100
600
Control
CaM
400
Ca2+i (nM)
200
250
500
750
1000

13. Regulation of Calcium release neuronal Calcium Binding Proteins (CaBP)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. Bootman
In preparation

14. CaM or other CaM-like Ca2+ sensor proteins ?
Adapted from Haeseleer et al., 2000
Inhibitory
Activatory ? (Yang et al., 2002)

15. CaBP binds to the InsP3R CaBP GST 1-604 1-225 226-604 NH2 COOH ER CYT
IP3 binding core ( )
CaBP
GST
1-604
1-225

16. CaBP binds to a similar region of the InsP3R as CaM1
159
CaM
sCaBP1
+ Ca2+
-Ca2+/ EGTA A) B)
CaBP binds to a similar region of the InsP3R as CaM

17. Ratio of CaBP: peptide B
Binding of CaBP to the InsP3R is calcium independent
Ratio of CaBP: peptide B
CaBP
CaBP
1/10
1/1
1/2
1/3
1/4
1/5
1/6
1/8
+ Calcium
+ EGTA 2 4 6 8 10
0.0
0.5
1.0
Band intensity
Peptide B: CaBP

18. CaBP inhibits InsP3 binding to the InsP3R
4 µg Lbs-1
10 µM sCaBP1
5 µM Ca2+
Lbs-1
sCaBP1 Ca
Ca/
100
[3H]IP3 Binding (% vs control)
100%
78 ± 1.9 %
68 ± 6.9 %
39 ± 4.7% 75 50 25
CaBP inhibits InsP3 binding to the InsP3R

19. Both Long and Short CaBPs inhibit InsP3 induced Calcium release
Control
200
400
600
800
1000
SCaBP
LCaBP
0.5µM
1µM
100µM
ATP
Time (s)
Ca2+i (nM)
Both Long and Short CaBPs
inhibit InsP3 induced Calcium release

20. CaBP inhibits InsP3 induced Calcium
release independent of Calcium binding
EF1
EF2
EF3
EF4
1000
800
600
400
200
1500
500
Time (s)
Ca2+i (nM)
0.5 1
100
µM ATP
Control
100 50
% responsive cells
0.5 1
ATP
(mM)
CaBP134

21. 10 mM InsP3 ester Control SCaBP1 time (s)
CaBP overexpression inhibits InsP3 Ester induced Calcium release:
CaBP acts directly on the InsP3R
10 mM InsP3 ester
250
200
Control
SCaBP1
150
Ca2+i (nM)
100 50
200
400
600
800
1000
1200
time (s)

22. Summary CaBP binds on the N-terminal CaM-binding site
in a Ca2+-independent way
CaBP inhibits IP3 binding
CaBP inhibits IICR
CaBP activity is Calcium independent

23. ??
GST
Control
IP3
AdPhos
CaM
sCaBP-1
1-225

24. Suramin Interacts with the CaM-binding sites on the IP3R1
Control
100 µM suramin
IP3R1
Input
Seph
CaM-Seph
+ Suramin
EGTA
Ca 2+
Control
10 µM CaM
1-225
EGTA
Ca 2+

25. MLCK peptide inhibits IICR
High CaM affinity binding properties
IP3

26. Properties of the inhibition of IICR by MLCK peptide
Inhibition of IICR by other CaM-binding peptides?
RyR pep: 10 nM
IP3
Trp3C14: 100 nM
IP3

27. Summary Suramin interacts with the CaM-binding sites on the IP3R
Suramin Inhibits IICR by lowering the IC50 for
activation by IP3
MLCK peptide inhibits IICR by lowering Vmax
By removing endogenous CaM?
By disrupting intermolecular protein-protein
interaction?

28. Conclusions Ca2+ is the major regulator of the IP3R
CaM inhibts IICR only in the presence of Ca2+
Two CaM-binding sites on the IP3R
Ca2+ dependent in the regulatory domain
Ca2+ independent in the N-terminus
CaM can play an important role in intramolecular
protein-protein interactions within IP3R, as
removing endogenous CaM inhibits IICR

29. 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

30. 45Ca2+-efflux technique on permeabilized A7r5 cells
Culture medium
Saponin
45Ca2+ TG
SDS
Fractional loss

31. Ca2+ -induced Ca2+ Release in A7r5 cells
Ca2+ release vs control
CICR is not mediated via IP3R or RyR
Control
Heparin
2-APB
XeC
RuRed
Ryanodine

32. Characteristics of the CICR mode
Ca2+ dependence:
EC50 = 700 nM
Hill = 1.9
Mg2+ inhibition: EC50= 0.6 mM
ATP stimulation: EC50= 320 µM

33. Effects of CaM, CaM1 and CaM123440 30 20
control 10 10 20
Time (min)
control
CaM1
CaM
CaM1234

34. Calmodulin
Calmodulin
Calmodulin
Calmodulin1
Calmodulin1
Calmodulin1234
Long CaBP1
NCS -
1/Frequenin
Short CaBP1
1/FrequeninE120Q
Calmodulin1234
Calmodulin1234
Long CaBP1
Long CaBP1
Short CaBP1
Short CaBP1
NCS -
1/Frequenin
NCS
NCS - -
1/Frequenin
1/Frequenin
NCS
NCS - -
1/FrequeninE120Q
1/FrequeninE120Q

35. Preincubation with a CaM-binding peptide inhibits CICR
control
RyR CaM-BS
(peptide aa )
Ca2+ (3 µM)

36. CaM but not CaM1234 can restore CICR
Preincubation with
RyR CaM-BS
(peptide aa )
Ca2+ (3 µM)

37. CaM but not CaM1234 can restore CICR
Preincubation with
RyR CaM-BS
(peptide aa )
CaM
CaM1234
Ca2+ (3 µM)

38. Summary Novel CICR mechanism in A7r5 cells IC50: 700 nM
Activated by ATP
Inhibited by MgCl2
CICR mechanism is regulated by CaM

39. 224
576
PIP2
IP3 Gα
PLC
2749
2275

40. 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
Baylor College of Medicine, Houston, Texas
Andreas Jeromin