IP3-induced Ca2+ release and calmodulin

Laboratory of PhysiologyKULeuven

Leuven, Belgium

IP3R

I, II, III

AgonistsIP3 Ca2+

Intraluminal proteins: Chromogranins; Calnexin

Cytosolic proteins:

Calmodulin; CaBP

IRBIT

CARP

HAP1A-Htt

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

Calmodulin inhibits 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+

rele

ase

(% /

2min

)

CaM

Control

HBE cells

90% IP3R30

5

10

15

20

25

0 0.2 0.4 0.6 0.8 1 1.2

[Ca2+] (µM)

Ca2+

rele

ase

(% /

2min

)

Control

CaM

Lbs-1: IP3-

binding core (226-581)

aa 1-225

NH2

COOH

ER

CYT

Lbs-1

Lbs-1 1-225

1 581

W226 581

1-225kDa

62 –

49 –

38 –

28 –17 –14 –6 –

B

1 2 3 1 2 3

kDa

62 –

49 –

38 –

28 –17 –14 –6 –

B

1 2 3 1 2 3

Recombinant ligand-binding

domain of IP3R1 (LBS-1)

CaM at N-terminal site inhibits IP3 binding

CaM Adkins et. al., 2000

Ca2+CaMYamada et. al., 1995Lin et. al., 2000Ca2+CaMSII

control ca cam ca cam cam1234 ca cam12340

20

40

60

80

100

Ca2

+ C

aM12

34

10 µ

M

CaM

1234Ca2

+ C

aM

10 µ

M

apoC

aM5 µM

Ca

2+cont

rol

[3 H]I

P 3 bin

ding

(%

)Lbs-1His

Lbs-1 1-225His

1 581

W226 581

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]IP

3 bin

ding

(%)

[CaM1234] (µM)

EC50= 1.7µM

The inhibition is Ca2+ independent

A

B

C

E

D

F

1 1591-5-10 1-5-10

1-5-8-14 70% IQ (site1) 76% IQ

53% IQ

CaM-binding sites in the N-terminal region are Ca2+ independent

Ca2+ independent

EC50 1-1.5 µM

A B C D E F CaMCa2+A B C D E F CaM EGTA

10007505002500

600

400

200

0

0.5 1 100ATP (µM )

Ca2+

i (nM

) ControlCaM

In COS cells IICR is inhibited by CaM and by CaM1234

CaM1234

40

Ca2+

rele

ase

vs A

2318

7 (%

)

50

60

70

80

90100

0 1 2 3 4 5

control

CaM CaM1234

In permeabilized L15 cells IICR is inhibited by CaM and by CaM1234

L15 cells

0

25

50

75

100

IP3R1 IP3R3

CaM is not the Ca2+ sensor for the inhibition of IICR[Ca2+] µM

%

The Ca2+/CaM site in the coupling region is not involved in the CaM inhibition of IICR

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)

CONCLUSION:

The N-terminal Ca2+-independent CaM-binding site is responsible for the CaM inhibition of IICR

1) Interaction site for other CaM-like proteins ?

2) Involved in intramolecular interactions?

Significance ?

Adapted from Haeseleer et al., 2000

CaM or other CaM-like Ca2+ sensor proteins ?

Interaction with neuronal Calcium Binding Proteins (CaBP)

Inhibitory

Activatory ?(Yang et al., 2002)

CaBP1

GST GST1-581

GST1-225

GST226-581

CaBP1 binds to the N-terminal part of the IP3R

CaBP1 ?

NH2

COOH

ER

CYT

IP3 binding core (226-581)

aa 1-225

Ca2+CaM

CaM

A

B

C

E

D

F

1 159CaM CaM

sCaBP1 A BC D EF sCaBP1 A BC D EF

+ Ca2+ -Ca2+/ EGTA

A)

B)

CaBP1 binds to a similar region as CaM independently of Ca2+

Control

0 200 400 600 800 1000

1000

200

400

600

800

0

sCaBPlCaBP

0.5µM 1µM 100µMATP

Time (s)

Ca2+

i (nM

)Both long and short CaBPs

inhibit IP3-induced calcium release in COS-1 cells

0.01 0.1 1 10 100

0

20

40

60

80

control 10 µM sCaBP

Ca

2+ re

leas

e vs

A23

187

(%)

IP3 µM

45Ca2+ flux

CaBP inhibits IP3-induced Ca2+

release independent of Ca2+ binding

EF1 EF2 EF3 EF4

CaBP134

1000

800

600

400

200

0 1500500 1000Time (s)

0

[Ca2+

] i (nM

)

0.5 1 100µM ATP

Control

YFP

Significance ?

1) Interaction site for other CaM-like proteins ?

2) Involved in intramolecular interactions?

Suramin interacts with the CaM-binding sites on IP3R1

1-225

EGTA Ca 2+

IP3R1

Inpu

t

Seph

Ca M

- Se p

h

+ S

uram

in

Seph

Ca M

- Se p

h

+ S

uram

inEGTA Ca 2+

Control

10 µM CaM

0.01 0.1 1 10

0

10

20

30

40

50

Ca2+

rele

ase

vs A

2318

7 (%

)

[IP3] (µM)

Control

100 µM suramin

Suramin mimics the CaM inhibition of IICR

IP3 binding core (IBC)

NH2

COOH

ER

CYT1-->225

CaM

Ca 2+CaM

Interaction 1-225 with IBC

GST /

IP

3

A

dPho

s

CaM

sCaB

P-1

0

50

100

1 2 3 4 5

N-terminal CaM-binding site = intramolecular interaction site?

SuraminCABP1

CaM1234

In permeabilized A7r5 cells

Suramin induced a large IP3-independent Ca2+ release

0 2 4 6 8 10 12 14 16 185

1015202530354045505560657075808590

fract

iona

l los

s (%

/ 2

min

)

time (min)

0 µM suramin 10 µM suramin 33.3 µM suramin 100 µM suramin 333 µM suramin 10 µM A23187

0 2 4 6 8 10 12 14 16 18

10

15

20

25

30

35

time (min)

100 µM suramin 100 µM sur + heparin

0 2 4 6 8 10 12 14 16 18 200

10

20

30

40

50

60

70

Frac

tiona

l Los

s (%

/ 2 m

in)

Time (min)

3 µM Ca2+

1 µM IP3

A23187 3 µM Ca2+ + 10 µM CaM1234

New type of Ca2+ -induced Ca2+ release (CICR) channel ??

+ CaM1234

0

20

40

60

80

100

120

RYRuRedXeC2-APBHEPCTR

Frac

tiona

l los

s vs c

ontr

ol (%

)

0.1 1 10

0

5

10

15

20

25

30C

a2+ r

elea

se v

s A23

187

(%/2

min

)

[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 A23

187

(%/2

min

)

[Ca2+] µM

0 10 200

10

20

30

40

Frac

tiona

l los

s (%

/ 2 m

in)

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

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

+ re

leas

e vs

con

trol

(%)

[CaBP1] (µM)0.1 1 10

0

20

40

60

80

100

Ca2+

rel

ease

vs c

ontr

ol (%

)

[GST-NCS-1] (µM)

0 100

10

20

30

40

50Fr

actio

nal l

oss (

%/2

min

)

Time (min)

Ca2+ (3 µM)

control

RyR1 CaM-BS

(peptide aa 3614-3643)

Preincubation with a CaM-binding peptide inhibits CICR

CaM but not CaM1234 can restore CICR

Preincubation with

RyR1 CaM-BS

(peptide aa 3614-3643)

Ca2+ (3 µM)

0 100

10

20

30

40

50Fr

actio

nal l

oss (

%/2

min

)

Time (min)

CaM

0 100

10

20

30

40

50Fr

actio

nal l

oss (

%/2

min

)

Time (min)

CaM but not CaM1234 can restore CICR

Preincubation with

RyR1 CaM-BS

(peptide aa 3614-3643)

Ca2+ (3 µM)

CaM1234CaM

New type of Ca2+ -induced Ca2+ release channel ??

IP3R CICR

CaM

CONCLUSIONS

•New type of intracellular Ca2+ channel (Wissing et al, 2002)?•Related to polycystin-2 (Koulen et al., 2002)?•Related to TRPV1 (Liu et al., 2003)?•Truncated IP3R?

CICR channel

Ca2+

+

ATP

+

suramin+

CaM is the Ca2+ sensor

Mg2+

-

CaM1234

-Inhibited by CaM mutantsInhibited by CaM-like proteins

Jan B. PARYS

Geert CALLEWAERT

Ludwig MISSIAEN

Rafael A. FISSORE

Nael NADIF KASRI

Geert BULTYNCK

Karolina SZLUFCIK

Leen VERBERT

Elke VERMASSEN

Zerihun ASSEFA

Iris CARTON

Patrick DE SMET

Babraham InstituteCambridge UK

H. Llewelyn Roderick

Martin D. Bootman

Michael Berridge

KULeuvenLeuven, Belgium

Andreas Jeromin

Baylor College of MedicineHouston, Texas

The Division of Molecular NeurobiologyThe Institute of Medical ScienceThe University of Tokyo

Katsuhiko Mikoshiba