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Calmodulin in action: Diversity in target recognition and Activation Mechanism
VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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
EF-hand motifs of CaM
closed open
apoCaM Ca2+CaM
Alpha helix
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!!
Calomodulin binding to target peptides
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
VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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
VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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
Mutation in IQ motif reduce or eliminate CDI
Calmodulin supports both inactivation and facilitation of L-type calcium channels
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
Lobe specific regulation by Calmodulin
Wt CaM enhances CDI
CaM1234 inhibits CDI
CaM12 enhances CDI
CaM34 inhibits CDI
Pitt et al., 2001
Possible models for CDI of L-type Ca2+ channels
Erickson et al., 2003
Active site remodeling
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
R-Type channel
L-Type channel
CaM mediates R-and N-Type channel CDI, via the interaction of the N-lobe CaM
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
VGCC LGCC SOCC
MSCC
PMCA
NCX
IP3R
Golgi
SPCA1
MitochondriaNCX
Uniporter
IP3R RYR
ER/SR
SERCA
Others??
R
Second messenger
CaM
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
C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free CaM functions as an agonist
C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free CaM functions as an antagonist
C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ free CaM functions as an agonist
C
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+ C-lobe Movement driven by Ca2+
N
C1C2
N2 N1
Lobe dependent regulation of RyR1 by calmodulin
Ca2+
C
CaM functions as an inhibitor
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
Binding of apocaM and Ca2+CaM to the RyR1
NMDA receptors: Lobe dependent interaction of CaM
SK channels: voltage independent and activated by submicromolar intra Ca2+
K+
Calmodulin-Induced Ion Channel Dimerization
Ca 2+CaM
Ca2+ permeant channels
CaM
RyRCaMLow Ca 2+
CaM
K+ channels PMCA
CaM
Ca2+ permeant channels
CaM
RyRHigh Ca 2+
K+ channels PMCA
CaM
CaM CaM
Ca2+ permeant channels
CaM
RyRHigh Ca 2+
K+ channels PMCA
CaM
CaM CaM
Calmodulin: Mediator of the calcium Modulation of Multiple Ion Channels
Calcium-Binding Protiens: Intracellular sensors from the Calmodulin Superfamily
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
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….
CaBP: intracellular sensors for the calmodulin superfamily
Adapted from Haeseleer et al., 2002
Extra helical turn
CaBPs are myristoylated and targeted to the membrane
sCaBP1 lCaBP1
control GFP
Haeseleer et al. 2000
Recoverin: member of the Neuronal calcium sensors
Ca 2+ -induced myristoyl switch
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
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
)
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
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
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
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
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
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)
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
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
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
Preassociation of apo-cam with various ion cahnnels controlled by Ca2+CaM is critical for a swift and potent respons
Conclusions
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