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