Intracellular calcium release channels as multi-protein complexes Jan B. Parys K.U.Leuven 7 th ECS...

Intracellular calcium release channels as multi-protein

complexes

Jan B. ParysK.U.Leuven

7th ECS meeting, June 13th, 2002

Intracellular Ca2+ release channels: IP3Rs and RyRs

(Furuichi & Mikoshiba, 1995)

IP3R1IP3R2IP3R3

RyR1RyR2RyR3

Intraluminal proteins

CalreticulinCalsequestrin

Chromogranins A and B

Annexin VIER

membrane proteins

Triadin

Junctin

Kinase anchor proteins

Plasma membrane proteins

Trp’s DHPR

G-proteinsCytosolic proteins

CaM CaBPs

FKBP’s

SorcinS100

IRAG

Cytoskeletal proteinsHomer

Ankyrin

TalinVinculin

Alpha-actin

Myosin II

1) What is the relation between the IP3R and the FKBP-type immunophilins ?

2) What is the relation between the IP3R and calmodulin ?

3) What is the relation between the IP3R and the cytoskeletal elements ?

FKBP12 and FKBP12.6

• 12-kDa FK506-binding protein

• Peptidylprolyl isomerase activity ( FK506)

• Immunophilins (+FK506) inhibit calcineurin

• Modulators of the IP3 receptors ?

Stabilization of the channelPromotion of coupled gating

• Modulators of the ryanodine receptors ( FK506)

Huse et al., 1999

FKBP12-binding

site

IP3R1 VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP3R2 ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP3R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV

RyR1 QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2 HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3 QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL

Y2H

FKBP12-binding

siteIP3

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+

Ca2+Ca2+

FKBP12

NH2

COOH

SI

SIIPORE

SIII

P PATP

ATP

IP3R1 VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP3R2 ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP3R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV

RyR1 QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2 HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3 QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL

Cytosol

Lumen of the store

FK506 and FKBP12 affect Ca2+ releasethrough the RyRs but not through the IP3Rs

0

200

400

600

800

1000

1200

1 min

FK50

6

Caf

fein

e

[Ca

2+] (

nM)

BC3H1

100

150

200

250

300

350

FK50

6

1 min

IP3

[Ca

2+] (

nM)

SH-SY5Y

Micr

osom

esSo

lubi

lizat

eG

STG

ST-F

KBP1

2 Retention of the intracellular Ca2+-release channels by GST-

FKBP12 affinity assay

RyR3 (HEK293)

RyR1 (Skel. muscle)

Cerebellum:

IP3R1

RyR1

A7r5:

IP3R1

IP3R3

Sf9: IP3R1

IP3R3

RyR3 IRIRSILRSLVPTEDLVGIISIP

RyR3/V2322L IRIRSILRSLLPTEDLVGIISIP (as in IP3R1) RyR3/V2322I IRIRSILRSLIPTEDLVGIISIP (as in RyR2) RyR3/V2322D IRIRSILRSLDPTEDLVGIISIP (negat. charge) RyR3/IP3R1 IRIRSICNSLLPLDDIVGIISIP (as in IP3R1)

Mutational analysis of FKBP12-binding site

0

5

10

15

20

25

30

35

40

Boun

d (%

)

GST-FKBP12 GST-FKBP12.6

2445-GKGEALRIRAILRSLVPLEDLVGIISLPLQIP

2412-GKGEAIRIRSILRSLIPLGDLVGVISIAFQMP

2309-GKGEAIRIRSILRSLVPTEDLVGIISIPLKLP

hum an R yR 1

hum an R yR 2

hum an R yR 3

1387-TEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIP

1386-TEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIP

1309-AEGKNVYTEIKCTSLVPLEDVVSVVTHEDCIT

hum an IP R 13

hum an IP R 23

hum an IP R 33

2307-GKGEAIRIRSILRSL PTEDLVGIISIPLKLP



2307-GKGEAIRIRSI GIISIPLKLP

L

I

D

CNSLLPLDDIV

R yR3/V2322L

R yR3/V2322 I

R yR3/V2322D

R yR3/IP R1 2318-2328

3

FKBP12 : binding protein

binding stable interaction

RyRs : XP residue as an -helix breaker

tw isted-am ide transition sta te

cis con figu ration

trans con figu rationstab ilized

in te rm ediate s ta te

Free

ene

rgy,

G

R eac tion coord ina te

RyR1

w ithout FK BP 12w ith FK BP 12

(valylpro lyl)

FKBP12 : enzyme

catalysis transient interaction

IP3Rs : XP residue in a different secondary structure

tw is ted-am ide tra nsition s ta te

cis con figu ration

trans con figurationstab ilized

in te rm ediate sta teFr

ee e

nerg

y, G

R eac tion coord ina te

IP R13

w ithout FK BP 12w ith FK BP 12

(leucylpro ly l)

Putative model

RyRs and IP3Rs have differentFKBP-binding properties

FKBP12-binding site on the IP3R is much less efficient than the homologous site in the RyR.

Higher-order structure may be important. RyRs: stable interaction

FKBP12 is regulatory subunit. IP3Rs: weak or transient interaction

chaperone function? effects on kinetics?

FK506 can act on SERCA and on calcineurin.

Effects on RyR

(Rodney et al., 2001)

3614 3643

ApoCaM: Ca2+/CaM:

ACTIVATOR OF RYR1 INHIBITOR OF RYR1

Ca2+

http://www.jbc.org/content/vol276/issue3/images/large/bc0219781008.jpeg

IP3R

A7r5 (Missiaen et al., 1999)

0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rele

ase

(% /

2min

)

Effects of CaM on Ca2+ release:

IP3R


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rele

ase

(% /

2min

)

Cerebellum (Michikawa et al., 1999)

200 μM Ca2+ +10 μM CaM +20 μM CaM


http://www.neuron.org/content/vol23/issue4/images/large/NEURO.23_4_799.9045.f3.jpeg

IP3R


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rele

ase

(% /

2min

)



Sf9 (Cardy & Taylor, 1998)


Effects of CaM on IP3 binding:


IP3R


0

20

40

60

- CaM + CaM

Free [Ca2+] (µM) 0.60.30.10.03<0.001

Ca2+

rele

ase

(% /

2min

)



Sf9 (Cardy & Taylor, 1998)

[3H

]IP

3 b

ind

ing

(%

)

0

20

40

60

80

100

120

* * ****

*

0 10

*

0 0.3 1 3 10 0 0.3 1 3 10


Effects of CaM on IP3 binding:

CaM (μM)

Lbs-domains (Vanlingen et al., 2000)

Lbs-1 Lbs-2 Lbs-3


IP3

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+

Ca2+Ca2+

CaM

CaMNH2

COOH

SI

SII CaM

PORE

SIII

Cytosol

Lumen of the store

P PATP

ATP

CaM-binding

sitesHigh-affinityCa2+-dependentTypes 1 and 2

Low-affinityCa2+-dependentNot in neuronalIP3R1 (SII+)

control ca cam ca cam cam1234 ca cam12340

20

40

60

80

100

Ca2+ C

aM12

34

CaM

1234

Ca2+

CaM

ApoC

aM

Ca2+

Cont

rol

[3 H]IP

3 bin

ding

(%

)

0.1 1 100

20

40

60

80

100

[3 H]IP

3 bin

ding

(%

)

Lbs-1

Lbs-1 1-225

1

581226

581+HIS

+HIS

Effects of CaMon IP3 binding

CaM1234 (μM)

control ca cam ca cam cam1234 ca cam12340

20

40

60

80

100

Ca2+ C

aM12

34

CaM

1234

Ca2+

CaM

ApoC

aM

Ca2+

Cont

rol

[3 H]IP

3 bin

ding

(%

)

0.1 1 100

20

40

60

80

100

[3 H]IP

3 bin

ding

(%

)

Lbs-1

Lbs-1 1-225

1

581226

581+HIS

+HIS

Effects of CaMon IP3 binding

CaM1234 (μM)

IC50 ~ 2 μM

Cyt1 Cyt2

Lbs-1

Lbs-1 1-225

1

581226

581

1 159

154 309

+HIS

+HIS

+GST

Ca2+

EGTA

Detailed localisation of the N-terminal CaM-binding site

CaM1234

Cyt1 Cyt2

Lbs-1

Lbs-1 1-225

1

581226

581

1 159

154

+HIS

+HIS

+GST

A

B

C

E

D

F

1-5-10 1-5-101-5-8-14 70% IQ 76% IQ

53% IQ


A B C D E F-0.20.00.20.40.60.81.0

200 µM free Ca 2+

1 mM EGTA

309

• Band-shift experiments on non-denaturing gels

• Interaction with dansyl-CaM

Cyt1 Cyt2

Lbs-1

Lbs-1 1-225

1

581226

581

1 159

154

+HIS

+HIS

+GST

A

B

C

E

D

F

1-5-10 1-5-101-5-8-14 70% IQ 76% IQ

53% IQ


A B C D E F-0.20.00.20.40.60.81.0

200 µM Ca2+

1mM EGTA

309

Inte

nsity

loss

(1-B

/Bo)

Kd 0.1 μM Kd 1 μM

0102030405060708090

100

0.01 0.03 0.1 0.3 0.6 1

[Ca2+] (µM)

Ca

2+ r

elea

se (%

/ 2 m

in)

1 µM IP3

1 µM IP3/ 10 µM CaM

1 µM IP3/ 10 µM CaM1234

CaM1234 does not inhibit IP3-induced Ca2+ release

IP3 1 μM

IP3 1 μM + CaM 10 μM

IP3 1 μM + CaM1234 10 μM

Discontinuous (aa 49-81 and 106-128).

Low affinity.

Ca2+ independent.

Involved in inhibition of IP3 binding.

Not involved in inhibition of IP3-induced Ca2+ release.

Other possible functions:Role in conformation of the IP3R ?Tethering of CaM ?Binding site for CaM-like proteins ? CaBP-1?Protective effect ? Proteolysis? Oxidative stress?

N-terminal CaM-binding site of the IP3R

Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells

IP3R1 IP3R3

Redistribution of IP3R1 after prolonged stimulation

Resting cells + AVP

AVP > 1hImipramineIP3-ester ThapsigarginCPA

[Ca2+]cyt

0

20

40

60

80

100

Role of PKC perinuclear IP3R1 cytoplasmic IP3R1

Percentages of cells with:C

ells

(%)

Con

trol

Con

trol

AVP

AVP

AVP+

Stau

r.

AVP+

Stau

r.

TG+S

taur

.

TG+S

taur

.

AVP+

BIM

AVP+

BIM

OAG

OAG

Role of cytoskeleton

0

10

20

30

40

50

60

70

80

Cel

ls (%

)

perinuclear IP3R1 cytoplasmic IP3R1

Percentages of cells with:C

ontro

lAV

P+N

ocod

.

AVP

Con

trol

AVP+

Noc

od.

AVP

Resting cells

+AVP

Microtubular network

Determinants of the localization of IP3Rs

Various IP3R isoforms can have a different distribution.

The localization of the IP3Rs is dynamic and change with the physiological status of the cell.

PKC activation seems an important determinant of the intracellular localization pattern.

The microtubular network is involved but which protein(s) determine the IP3R localization is not yet ascertained.

Conclusions The intracellular Ca2+ release channels IP3Rs and RyRs act as part of a multiprotein complex. Many proteins have been proposed as partners. The existence of consensus binding sites is not sufficient for assuring binding in vivo. Higher-order structure, experimental conditions and indirect associations can be involved. RyRs and IP3Rs are regulated by different proteins.

Future work on the IP3R will focus on the interactions with the various Ca2+-binding proteins and with the different scaffolding proteins.

Geert Callewaert

Humbert De Smedt

Ludwig Missiaen

Jan B. Parys

IP3-team (Leuven, Belgium)Geert Bultynck

Patrick De Smet

Nael Nadif Kasri

Ilse Sienaert

Karolina Szlufcik

Kristel Van Acker

Sara Vanlingen

Elke Vermassen

Intracellular calcium release channels as multi-protein complexes Jan B. Parys K.U.Leuven 7 th ECS...

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