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Calmodulin in action: Diversity in target recognition and Activation Mechanism
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VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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Calmodulin: a Prototypical Calcium Sensor
Ca2+
Ca2+ buffers
Ca2+ sensors
Effectors
Calbindin,parvalbumin
Calmodulin
2. Molecular level: promoting different modes of association with many targetmolecules
1. Cellular level: subcellular distribution
3. Conformational state of CaM: target specificity
Regulation of CaM
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EF-hand motifs of CaM
closed open
apoCaM Ca2+CaM
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Alpha helix
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CaM-binding sites
Ca2+ CaM-binding sites:
Amphiphatic alpha-helices,20 residuesBasic and hydrophobic residuesAromatic residue at N-terminusBaa motif: Type A: 1-5-10 (FILVW)xxx(FILV)xxxx(FILVW)Type B: 1-8-14 (FILVW)xxxxxx(FAILVW)xxxxx(FILVW)
apoCaM-binding sites: IQ motifs: IQXXXRGXXR [ILV]QXXXRXXX[RK]
CaM-binding sites are not conserved!!
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Calomodulin binding to target peptides
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Low Ca2+ High Ca2+Mechanism by which CaM regulate their target
1Phosphorylase kinase
neuromodulin, neurogranin
2
3
MLCK, calcineurin
4IP3R
6
5CaMKI, II, IV
CaMK II
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VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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PMCA pump: Relieve autoinhibition
Ca2+ CaM
N N
C
C
Autoinhibited Activated
CaM is the main regulator of the pump.
CaM increases affinity for Ca2+ and the Vmax
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VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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Modulation of Voltage gated Ca2+ channels by calmodulin
L-typeP/Q typeR-typeN-typeT-type
Calmodulin mediates both: CDF and CDI
L
R
P/QN
T
α1-,β-, α2δ and sometimes a γ subunit
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Mutation in IQ motif reduce or eliminate CDI
Calmodulin supports both inactivation and facilitation of L-type calcium channels
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1. CaM1234 inhibits CDI2. CaM binds to IQ motif3. Mutation in IQ motif reduce or eliminate CDI4. Faciliation is blocked by CaM1234
Calmodulin is tethered to the L-type Ca 2+ channel
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Lobe specific regulation by Calmodulin
Wt CaM enhances CDI
CaM1234 inhibits CDI
CaM12 enhances CDI
CaM34 inhibits CDI
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Pitt et al., 2001
Possible models for CDI of L-type Ca2+ channels
Erickson et al., 2003
Active site remodeling
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Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ ChannelsCaM facilitates opening
CaM enhances CDI
CResting
Facilitated inactivated
C
N
C
Normal openC
N
C
Facilitated open
C
N
C
Normal inactivated
C
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R-Type channel
L-Type channel
CaM mediates R-and N-Type channel CDI, via the interaction of the N-lobe CaM
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C N CN
C
N
IQ
preIQ
EF + N C
R-Type
P/Q-TypeN-Type
L-TypeN C
Not found
Not found
Not foundCDICDI
CDI
CDI
CDF
Buffer sensitive Buffer insensitiveDetects global Ca 2+ entry Detects local Ca 2+ entry
Fast VDI
slow VDI
(N-lobe)modulated
(C-lobe)modulated
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VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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Ryanodine receptor 1
apoCaM activates RyR1
Ca2+ CaM inactivates RyR1
Calcium binding Leads to an N-terminal shift in Its binding site on the RyR
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C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free CaM functions as an agonist
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C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free CaM functions as an antagonist
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C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free CaM functions as an agonist
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C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ C-lobe Movement driven by Ca2+
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N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+
C
CaM functions as an inhibitor
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C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free
Agonist
C
N
C1C2
N2 N1Ca2+
C
N
C1C2
N2 N1
C
N
C1C2
N2 N1
Ca2+ Inhibitor
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Binding of apocaM and Ca2+CaM to the RyR1
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NMDA receptors: Lobe dependent interaction of CaM
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SK channels: voltage independent and activated by submicromolar intra Ca2+
K+
Calmodulin-Induced Ion Channel Dimerization
Ca 2+CaM
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Ca2+ permeant channels
CaM
RyRCaMLow Ca 2+
CaM
K+ channels PMCA
CaM
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Ca2+ permeant channels
CaM
RyRHigh Ca 2+
K+ channels PMCA
CaM
CaM CaM
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Ca2+ permeant channels
CaM
RyRHigh Ca 2+
K+ channels PMCA
CaM
CaM CaM
Calmodulin: Mediator of the calcium Modulation of Multiple Ion Channels
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Calcium-Binding Protiens: Intracellular sensors from the Calmodulin Superfamily
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CaM
CaM-like
L-CaBP1
S-CaBP1
Caldendrin
S-CaBP5
S-CaBP2
L-CaBP2
S-CaBP1
CaBP3
GCAP
GCAP3
GCAP2
Recoverin
Visinin
Neurocalcin
VILIP3
VILIP1
VILIP2
NCS1
EF1 EF2 EF3 EF4
CaM
EF1 EF2 EF3 EF4
CaBP7-8
EF1 EF2 EF3 EF4
Recoverin
EF1 EF2 EF3 EF4
NCS1
Adapted from Haeseleer et al., 2000
EF3 EF4
GCAPs
EF2EF1
EF1 EF2 EF3 EF4
CaBP1-5
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Exocytosis inEndocrine cells
Exocytosis inSynapses
Channelregulation
Basal Ca2+
NCS1 VILIP-1 CaM Synaptotagmin
7 6 5 4 3
100
80
60
40
20
0
Ca2+
bo
un
d
-log[Ca2]+ Adapted from Burgoyne and Weiss 2001
Why so many CaBPs….?
Cellular Localisation and….
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CaBP: intracellular sensors for the calmodulin superfamily
Adapted from Haeseleer et al., 2002
Extra helical turn
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CaBPs are myristoylated and targeted to the membrane
sCaBP1 lCaBP1
control GFP
Haeseleer et al. 2000
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Recoverin: member of the Neuronal calcium sensors
Ca 2+ -induced myristoyl switch
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Regulation of P/Q-type voltage dependent Ca2+ channel by CaBP1
Ca2+/Calmodulin dependent inactivation and facilitation.
CaBP1 enhances inactivation and does not support CDF
NCS1 was shown to mediate a rapid ca2+ dependent Facilitation P/Q-type Ca2+ channel and enhances inactivationCa2+ independent
Only inhibitory action
Ca2+-independent
Why different: different affinities for Ca2+Calmodulin and CaBP1 are direcly bound to the channel
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New type of Ca2+ -induced Ca2+ release (CICR) mechanisme in A7r5 cells
0
20
40
60
80
100
120
RYRuRedXeC2-APBHEPCTR
Frac
tiona
l los
s vs c
ontr
ol (%
)
0 20100
50
100
45Ca2+ -flux on permeabilized A7r5 cells
ER ER
Intact Permeabilized 45Ca2+ Loaded
ER
IP3
Ionophore
Ca2+
Fra
ctio
nal l
oss
(%/2
min
)
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0.1 1 10
0
5
10
15
20
25
30
Ca2+
rel
ease
vs
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
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0.1 1 10
0
20
40
60
80
100
Ca
2+ r
elea
se v
s con
trol
(%)
[CaM] (µM)
Effects of CaM and CaM1234 on CICRF
ract
iona
l los
s (%
/ 2 m
in)
Time (min)
0 10 20
0
10
20
30
40
CaM1234control
CaM
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0.1 1 10
0
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)
EF1 EF2 EF3 EF4
CaM
EF1 EF2 EF3 EF4
CaBP1 short/long
EF1 EF2 EF3 EF4
NCS1
EF1 EF2 EF3 EF4
NCS1 3
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0 100
10
20
30
40
50F
ract
ion
al lo
ss (
%/2
min
)
Time (min)
Ca2+
control
RyR1 CaM-BS
(peptide aa 3614-3643)
Preincubation with a CaM-binding peptide inhibits CICR
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0 10
0
5
10
15
20
25
30
35
40
45
50
Fra
ctio
nal l
oss
(%/2
min
)
Time (min)
CaM but not CaM1234 can restore CICR
Preincubation with
RyR1 CaM-BS
(peptide aa 3614-3643)
Ca2+
CaM but not CaM1234 can restore CICR
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0 10
0
5
10
15
20
25
30
35
40
45
50
Fra
ctio
nal l
oss
(%/2
min
)
Time (min)
CaM but not CaM1234 can restore CICR
Ca2+
CaM
Preincubation with
RyR1 CaM-BS
(peptide aa 3614-3643)
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0 10
0
5
10
15
20
25
30
35
40
45
50
Fra
ctio
nal l
oss
(%/2
min
)
Time (min)
Preincubation with
RyR1 CaM-BS
(peptide aa 3614-3643)
Ca2+
CaM
CaM1234
CaM but not CaM1234 can restore CICR
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In permeabilized A7r5 cells
Suramin induced a large IP3-independent Ca2+ release
0 2 4 6 8 10 12 14 16 18
10
20
30
40
50
60
70
80
90
frac
tiona
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
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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
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Preassociation of apo-cam with various ion cahnnels controlled by Ca2+CaM is critical for a swift and potent respons
Conclusions