Intracellular calcium release channels as multi protein complexes
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Intracellular calcium release channels as multi-protein complexes. Jan B. Parys K.U.Leuven. 7 th ECS meeting, June 13th, 2002. Intracellular Ca 2+ release channels: IP 3 Rs and RyRs. (Furuichi & Mikoshiba, 1995). Plasma membrane proteins. Cytoskeletal proteins. Homer. DHPR. Trp’s.

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Intracellular calcium release channels as multi protein complexes

Intracellular calcium release channels as multi-protein complexes

Jan B. Parys

K.U.Leuven

7th ECS meeting, June 13th, 2002


Intracellular calcium release channels as multi protein complexes

Intracellular Ca2+ release channels: IP3Rs and RyRs

(Furuichi & Mikoshiba, 1995)


Intracellular calcium release channels as multi protein complexes

Plasma membrane proteins

Cytoskeletal proteins

Homer

DHPR

Trp’s

Ankyrin

Vinculin

Talin

Cytosolic proteins

G-proteins

Alpha-actin

Myosin II

CaM

CaBPs

Sorcin

S100

Kinase anchor proteins

IRAG

Calsequestrin

Triadin

Calreticulin

FKBP’s

Junctin

Annexin VI

ERmembrane proteins

Chromogranins A and B

Intraluminal proteins

IP3R1IP3R2IP3R3

RyR1RyR2RyR3


Intracellular calcium release channels as multi protein complexes

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

  • What is the relation between the IP3R and calmodulin ?

  • What is the relation between the IP3R and the cytoskeletal elements ?


Intracellular calcium release channels as multi protein complexes

FKBP12 and FKBP12.6

  • 12-kDa FK506-binding protein

  • Peptidylprolyl isomerase activity ( FK506)

  • Immunophilins (+FK506) inhibit calcineurin

Huse et al., 1999

  • Modulators of the ryanodine receptors ( FK506)

Stabilization of the channel

Promotion of coupled gating

  • Modulators of the IP3 receptors ?


Intracellular calcium release channels as multi protein complexes

FKBP12-binding site

Y2H

IP3R1VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV

IP3R2ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV

IP3R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV

RyR1QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG

RyR2HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI

RyR3QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL


Intracellular calcium release channels as multi protein complexes

SI

FKBP12-binding site

Ca2+

NH2

IP3

Ca2+

SIII

Ca2+

Ca2+

FKBP12

Ca2+

Ca2+

P

P

ATP

COOH

ATP

Ca2+

SII

PORE

Cytosol

Lumen of the store

IP3R1VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV

IP3R2ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV

IP3R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV

RyR1QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG

RyR2HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI

RyR3QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL

Ca2+


Intracellular calcium release channels as multi protein complexes

SH-SY5Y

BC3H1

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


Intracellular calcium release channels as multi protein complexes

A7r5:

IP3R1

IP3R3

Sf9:

IP3R1

IP3R3

Cerebellum:

IP3R1

RyR1

Retention of the intracellular Ca2+-release channels by GST-FKBP12 affinity assay

2

1

s

e

e

P

t

a

B

m

z

K

o

i

l

s

i

F

b

-

o

T

T

r

u

l

c

S

S

i

o

M

G

G

S

RyR1 (Skel. muscle)

RyR3 (HEK293)


Intracellular calcium release channels as multi protein complexes

Mutational analysis of FKBP12-binding site

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)


Intracellular calcium release channels as multi protein complexes

Putative model

RyRs : XP residue as an

-helix breaker

IP3Rs : XP residue in a different secondary structure

FKBP12 : binding protein

binding  stable interaction

FKBP12 : enzyme

catalysis  transient interaction


Intracellular calcium release channels as multi protein complexes

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.


Intracellular calcium release channels as multi protein complexes

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

  • What is the relation between the IP3R and calmodulin ?

  • What is the relation between the IP3R and the cytoskeletal elements ?


Intracellular calcium release channels as multi protein complexes

ApoCaM: Ca2+/CaM:

ACTIVATOR OF RYR1INHIBITOR OF RYR1

Effects on RyR

3614 3643

Ca2+

(Rodney et al., 2001)


Intracellular calcium release channels as multi protein complexes

IP3R

Effects of CaM on Ca2+ release:

A7r5 (Missiaen et al., 1999)


Intracellular calcium release channels as multi protein complexes

IP3R

Effects of CaM on Ca2+ release:

200 μM Ca2++10 μM CaM +20 μM CaM

A7r5 (Missiaen et al., 1999)

Cerebellum (Michikawa et al., 1999)


Intracellular calcium release channels as multi protein complexes

IP3R

Effects of CaM on Ca2+ release:

Effects of CaM on IP3 binding:

200 μM Ca2++10 μM CaM +20 μM CaM

A7r5 (Missiaen et al., 1999)

Cerebellum (Michikawa et al., 1999)

Sf9 (Cardy & Taylor, 1998)


Intracellular calcium release channels as multi protein complexes

IP3R

Effects of CaM on Ca2+ release:

Effects of CaM on IP3 binding:

200 μM Ca2++10 μM CaM +20 μM CaM

A7r5 (Missiaen et al., 1999)

Cerebellum (Michikawa et al., 1999)

Lbs-1 Lbs-2 Lbs-3

CaM (μM)

Sf9 (Cardy & Taylor, 1998)

Lbs-domains (Vanlingen et al., 2000)


Intracellular calcium release channels as multi protein complexes

SI

CaM

Ca2+

NH2

IP3

Ca2+

SIII

Ca2+

Ca2+

Ca2+

Ca2+

CaM

P

P

ATP

CaM

COOH

ATP

Ca2+

SII

PORE

Cytosol

Lumen of the store

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

Ca2+

CaM-binding sites

High-affinityCa2+-dependentTypes 1 and 2


Intracellular calcium release channels as multi protein complexes

1

581

Lbs-1

226

581

Lbs-1 1-225

Effects of CaMon IP3 binding

+HIS

+HIS

100

80

60

[3H]IP3 binding (%)

[3H]IP3 binding (%)

40

20

0

0.1

1

10

CaM1234 (μM)

Ca2+

Control

ApoCaM

Ca2+ CaM

CaM1234

Ca2+ CaM1234


Intracellular calcium release channels as multi protein complexes

1

581

Lbs-1

226

581

Lbs-1 1-225

Effects of CaMon IP3 binding

+HIS

+HIS

100

80

60

[3H]IP3 binding (%)

[3H]IP3 binding (%)

IC50~ 2 μM

40

20

0

0.1

1

10

CaM1234 (μM)

Ca2+

Control

ApoCaM

Ca2+ CaM

CaM1234

Ca2+ CaM1234


Intracellular calcium release channels as multi protein complexes

1

581

Lbs-1

226

581

Lbs-1 1-225

Cyt1 Cyt2

Detailed localisation of the N-terminal CaM-binding site

+HIS

+HIS

+GST

1

159

154

309

Ca2+

CaM1234

EGTA


Intracellular calcium release channels as multi protein complexes

1

581

Lbs-1

226

581

Lbs-1 1-225

Cyt1 Cyt2

Detailed localisation of the N-terminal CaM-binding site

+HIS

+HIS

+GST

1

159

154

309

1-5-10

1-5-10

53% IQ

1-5-8-14

76% IQ

70% IQ

A

C

D

B

E

F

1.0

2+

200 µM free Ca

0.8

  • Band-shift experiments on non-denaturing gels

  • Interaction with dansyl-CaM

1 mM EGTA

0.6

0.4

0.2

0.0

-0.2

A

B

C

D

E

F


Intracellular calcium release channels as multi protein complexes

1

581

Lbs-1

226

581

Lbs-1 1-225

Cyt1 Cyt2

Detailed localisation of the N-terminal CaM-binding site

+HIS

+HIS

+GST

1

159

154

309

1-5-10

1-5-10

53% IQ

1-5-8-14

76% IQ

70% IQ

A

C

D

B

E

F

1.0

200 µM Ca2+1mM EGTA

Kd 0.1 μM Kd 1 μM

0.8

0.6

0.4

Intensity loss (1-B/Bo)

0.2

0.0

-0.2

A

B

C

D

E

F


Intracellular calcium release channels as multi protein complexes

CaM1234 does not inhibit IP3-induced Ca2+ release

IP3 1 μM

IP3 1 μM + CaM 10 μM

IP3 1 μM + CaM1234 10 μM


Intracellular calcium release channels as multi protein complexes

N-terminal CaM-binding site of the IP3R

  • 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?


Intracellular calcium release channels as multi protein complexes

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

  • What is the relation between the IP3R and calmodulin ?

  • What is the relation between the IP3R and the cytoskeletal elements ?


Localization of ip 3 r1 and ip 3 r3 in a7r5 smooth muscle cells

Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells

IP3R1

IP3R3


Redistribution of ip 3 r1 after prolonged stimulation

AVP > 1h

ImipramineIP3-ester ThapsigarginCPA

[Ca2+]cyt

Redistribution of IP3R1 after prolonged stimulation

Resting cells

+ AVP


Intracellular calcium release channels as multi protein complexes

Percentages of cells with:

perinuclear IP3R1 cytoplasmic IP3R1

AVP

AVP

OAG

OAG

Control

Control

TG+Staur.

TG+Staur.

AVP+Staur.

AVP+Staur.

AVP+BIM

AVP+BIM

Role of PKC

100

80

60

Cells (%)

40

20

0


Role of cytoskeleton

Role of cytoskeleton

Percentages of cells with:

Resting cells

perinuclear IP3R1cytoplasmic IP3R1

+AVP

AVP

AVP

Control

Control

AVP+Nocod.

AVP+Nocod.

Microtubular network

80

70

60

50

Cells (%)

40

30

20

10

0


Determinants of the localization of ip 3 rs

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.

Determinants of the localization of IP3Rs


Conclusions

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.


Intracellular calcium release channels as multi protein complexes

IP3-team (Leuven, Belgium)

Geert Bultynck

Patrick De Smet

Nael Nadif Kasri

Ilse Sienaert

Karolina Szlufcik

Kristel Van Acker

Sara Vanlingen

Elke Vermassen

Geert Callewaert

Humbert De Smedt

Ludwig Missiaen

Jan B. Parys


Intracellular calcium release channels as multi protein complexes

IP3-team (Leuven, Belgium)

Geert Bultynck

Patrick De Smet

Nael Nadif Kasri

Ilse Sienaert

Karolina Szlufcik

Kristel Van Acker

Sara Vanlingen

Elke Vermassen

Geert Callewaert

Humbert De Smedt

Ludwig Missiaen

Jan B. Parys


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