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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-GKGEAIRIRSILRSL PTEDLVGIISIPLKLP
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.
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 ?
Effects on RyR
(Rodney et al., 2001)
3614 3643
ApoCaM: Ca2+/CaM:
ACTIVATOR OF RYR1 INHIBITOR OF RYR1
Ca2+
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
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
)
Cerebellum (Michikawa et al., 1999)
200 μM Ca2+ +10 μM CaM +20 μM CaM
Effects of CaM on Ca2+ release:
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
)
Cerebellum (Michikawa et al., 1999)
200 μM Ca2+ +10 μM CaM +20 μM CaM
Sf9 (Cardy & Taylor, 1998)
Effects of CaM on Ca2+ release:
Effects of CaM on IP3 binding:
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
)
Cerebellum (Michikawa et al., 1999)
200 μM Ca2+ +10 μM CaM +20 μM CaM
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 Ca2+ release:
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
Detailed localisation of the N-terminal CaM-binding site
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
Detailed localisation of the N-terminal CaM-binding site
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
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 ?
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
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