Regulation of the IP3 Receptor by Ca2+ and Ca2+-Binding Proteins

Laboratory of Physiology

KULeuven

Leuven, Belgium

VGCC LGCC SOCC

MSCC

PMCA

NCX

IP3R RYR

ER/SR

SERCA

IP3R

golgi

PMR1

MitochondriaNCX

Uniporter

Others??

Buffers

R

Second messenger

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

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

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

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

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)

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

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

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

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)

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

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

Adapted from Haeseleer et al., 2000

CaM or other CaM-like Ca2+ sensor proteins ?

Inhibitory

Activatory ?(Yang et al., 2002)

CaBP

GST GST1-604

GST1-225

GST226-604

CaBP binds to the InsP3R

NH2

COOH

ER

CYT

IP3 binding core (226-604)

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

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

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

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

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)

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)

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

??

-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

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

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

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

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?

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

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

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

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

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

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

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)

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

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)

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

Summary

Novel CICR mechanism in A7r5 cells

CICR mechanism is regulated by CaM

IC50: 700 nMActivated by ATPInhibited by MgCl2

2749

2275

224 576

Gα PIP2

IP3

PLC PLC

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