Calmodulin in action: Diversity in target recognition and Activation Mechanism

VGCC LGCC SOCC MSCC PMCA NCX IP 3 R Golgi SPCA1 Mitochondria NCX Uniporter IP 3 RRYR ER/SR SERCA Others?? R Second messenger CaM

Calmodulin: a Prototypical Calcium Sensor Ca 2+ Ca 2+ buffers Ca 2+ sensors Effectors Calbindin, parvalbumin Calmodulin 2. Molecular level: promoting different modes of association with many target molecules 1. Cellular level: subcellular distribution 3. Conformational state of CaM: target specificity Regulation of CaM

EF-hand motifs of CaM closedopen apoCaM Ca 2+ CaM

Alpha helix

CaM-binding sites Ca 2+ CaM-binding sites: Amphiphatic alpha-helices, 20 residues Basic and hydrophobic residues Aromatic residue at N-terminus Baa motif: Type A: (FILVW)xxx(FILV)xxxx(FILVW) Type B: (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 Ca 2+ High Ca 2+ Mechanism by which CaM regulate their target1 Phosphorylase kinase neuromodulin, neurogranin 2 3 MLCK, calcineurin 4 IP 3 R 6 5 CaMKI, II, IV CaMK II

VGCC LGCC SOCC MSCC PMCA NCX IP 3 R Golgi SPCA1 Mitochondria NCX Uniporter IP 3 RRYR ER/SR SERCA Others?? R Second messenger CaM

PMCA pump: Relieve autoinhibition Ca 2+ CaM NN C C AutoinhibitedActivated CaM is the main regulator of the pump. CaM increases affinity for Ca2+ and the Vmax

VGCC LGCC SOCC MSCC PMCA NCX IP 3 R Golgi SPCA1 Mitochondria NCX Uniporter IP 3 RRYR ER/SR SERCA Others?? R Second messenger CaM

Modulation of Voltage gated Ca 2+ channels by calmodulin L-type P/Q type R-type N-type T-type Calmodulin mediates both: CDF and CDI L R P/Q N 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.CaM 1234 inhibits CDI 2.CaM binds to IQ motif 3.Mutation in IQ motif reduce or eliminate CDI 4.Faciliation is blocked by CaM 1234 Calmodulin is tethered to the L-type Ca 2+ channel

Lobe specific regulation by Calmodulin Wt CaM enhances CDI CaM 1234 inhibits CDI CaM 12 enhances CDI CaM 34 inhibits CDI

Pitt et al., 2001 Possible models for CDI of L-type Ca 2+ channels Erickson et al., 2003 Active site remodeling

Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca 2+ Channels CaM facilitates opening CaM enhances CDI C Resting Facilitated inactivated C N C Normal open C 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 C N C N IQ preIQ EF+ NC R-Type P/Q-Type N-Type L-Type NC Not found CDI CDF Buffer sensitiveBuffer insensitive Detects global Ca 2+ entryDetects local Ca 2+ entry Fast VDI slow VDI (N-lobe) modulated (C-lobe) modulated

VGCC LGCC SOCC MSCC PMCA NCX IP 3 R Golgi SPCA1 Mitochondria NCX Uniporter IP 3 RRYR ER/SR SERCA Others?? R Second messenger CaM

Ryanodine receptor 1 apoCaM activates RyR1 Ca 2+ CaM inactivates RyR1 Calcium binding Leads to an N-terminal shift in Its binding site on the RyR

C N C1C2 N2N1 Lobe dependent regulation of RyR1 by calmodulin Ca 2+ freeCaM functions as an agonist

C N C1C2 N2N1 Lobe dependent regulation of RyR1 by calmodulin Ca 2+ freeCaM functions as an antagonist

C N C1C2 N2N1 Lobe dependent regulation of RyR1 by calmodulin Ca 2+ freeCaM functions as an agonist

C N C1C2 N2N1 Lobe dependent regulation of RyR1 by calmodulin Ca 2+ C-lobe Movement driven by Ca 2+

N C1C2 N2N1 Lobe dependent regulation of RyR1 by calmodulin Ca 2+ C CaM functions as an inhibitor

C N C1C2 N2N1 Lobe dependent regulation of RyR1 by calmodulin Ca 2+ free Agonist C N C1C2 N2N1 Ca 2+ C N C1C2 N2N1 C N C1C2 N2N1 Ca 2+ Inhibitor

Binding of apocaM and Ca 2+ CaM to the RyR1

NMDA receptors: Lobe dependent interaction of CaM

SK channels: voltage independent and activated by submicromolar intra Ca2+ K+K+ Calmodulin-Induced Ion Channel Dimerization Ca 2+ CaM

Ca 2+ permeant channels CaM RyR CaM Low Ca 2+ CaM K+ channelsPMCA CaM

Ca 2+ permeant channels CaM RyR High Ca 2+ K+ channelsPMCA CaM

Ca 2+ permeant channels CaM RyR High Ca 2+ K+ channelsPMCA 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 EF1EF2EF3EF4 CaM EF1EF2EF3EF4 CaBP7-8 EF1EF2EF3EF4 Recoverin EF1EF2EF3EF4 NCS1 Adapted from Haeseleer et al., 2000 EF3EF4 GCAPs EF2EF1 EF2EF3EF4 CaBP1-5

Exocytosis in Endocrine cells Exocytosis in Synapses Channel regulation Basal Ca2+ NCS1VILIP-1CaMSynaptotagmin Ca 2+ bound -log[Ca 2]+ 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 sCaBP1lCaBP1 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 inactivation Ca2+ independent Only inhibitory action Ca2+-independent Why different: different affinities for Ca2+ Calmodulin and CaBP1 are direcly bound to the channel

New type of Ca 2+ -induced Ca 2+ release (CICR) mechanisme in A7r5 cells Ca 2+ -flux on permeabilized A7r5 cells ER Intact Permeabilized 45 Ca 2+ Loaded ER IP 3 Ionophore Ca 2+ Fractional loss (%/2 min)

Characteristics of the CICR mode Ca 2+ dependence: EC 50 = 700 nM Hill = 1.9 Mg 2+ inhibition: EC 50 = 0.6 mM ATP stimulation: EC 50 = 320 µM

Effects of CaM and CaM 1234 on CICR Fractional loss (%/ 2 min) Time (min) CaM 1234 control CaM

EF1EF2EF3EF4 CaM EF1EF2EF3EF4 CaBP1 short/long EF1EF2EF3EF4 NCS1 EF1EF2EF3EF4 NCS1 3

Ca 2+ control RyR1 CaM-BS (peptide aa ) Preincubation with a CaM-binding peptide inhibits CICR

CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa ) Ca 2+ CaM but not CaM1234 can restore CICR

Ca 2+ CaM Preincubation with RyR1 CaM-BS (peptide aa )

Preincubation with RyR1 CaM-BS (peptide aa ) Ca 2+ CaM CaM 1234 CaM but not CaM1234 can restore CICR

In permeabilized A7r5 cells Suramin induced a large IP 3 -independent Ca 2+ release

New type of Ca 2+ -induced Ca 2+ release channel ?? IP 3 RCICR CaM CONCLUSIONS New type of intracellular Ca 2+ channel (Wissing et al, 2002)? Related to polycystin-2 (Koulen et al., 2002)? Related to TRPV1 (Liu et al., 2003)? Truncated IP 3 R? CICR channel Ca 2+ + ATP + suramin + CaM is the Ca 2+ sensor Mg 2+ - CaM Inhibited by CaM mutants Inhibited 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