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Calmodulin in action: Diversity in target recognition and Activation Mechanism

Calmodulin in action: Diversity in target recognition and Activation Mechanism. LGCC. VGCC. SOCC. MSCC. R. NCX. Second messenger. NCX. Mitochondria. CaM. PMCA. IP 3 R. RYR. IP 3 R. SPCA1. Others??. Uniporter. Golgi. ER/SR. SERCA. Calmodulin: a Prototypical Calcium Sensor.

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Calmodulin in action: Diversity in target recognition and Activation Mechanism

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  1. Calmodulin in action: Diversity in target recognition and Activation Mechanism

  2. LGCC VGCC SOCC MSCC R NCX Second messenger NCX Mitochondria CaM PMCA IP3R RYR IP3R SPCA1 Others?? Uniporter Golgi ER/SR SERCA

  3. Calmodulin: a Prototypical Calcium Sensor Regulation of CaM Ca2+ 1. Cellular level: subcellular distribution Ca2+ buffers 2. Molecular level: promoting different modes of association with many target molecules Ca2+ sensors Calbindin, parvalbumin Calmodulin 3. Conformational state of CaM: target specificity Effectors

  4. EF-hand motifs of CaM Ca2+CaM apoCaM closed open

  5. Alpha helix

  6. CaM-binding sites Ca2+ CaM-binding sites: Amphiphatic alpha-helices, 20 residues Basic and hydrophobic residues Aromatic residue at N-terminus Baa 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!!

  7. Calomodulin binding to target peptides

  8. 1 Phosphorylase kinase 2 neuromodulin, neurogranin 3 MLCK, calcineurin 4 IP3R 5 CaMKI, II, IV 6 CaMK II Mechanism by which CaM regulate their target High Ca2+ Low Ca2+

  9. CaM LGCC VGCC SOCC MSCC R NCX Second messenger NCX Mitochondria PMCA IP3R RYR IP3R SPCA1 Others?? Uniporter Golgi ER/SR SERCA

  10. PMCA pump: Relieve autoinhibition C Ca2+ CaM C N N Autoinhibited Activated CaM is the main regulator of the pump. CaM increases affinity for Ca2+ and the Vmax

  11. CaM LGCC VGCC SOCC MSCC R NCX Second messenger NCX Mitochondria PMCA IP3R RYR IP3R SPCA1 Others?? Uniporter Golgi ER/SR SERCA

  12. L P/Q N R T Modulation of Voltage gated Ca2+ channels by calmodulin α1-,β-, α2δ and sometimes a γ subunit L-type P/Q type R-type N-type T-type Calmodulin mediates both: CDF and CDI

  13. Calmodulin supports both inactivation and facilitation of L-type calcium channels Mutation in IQ motif reduce or eliminate CDI

  14. CaM1234 inhibits CDI • CaM binds to IQ motif • Mutation in IQ motif reduce or eliminate CDI • Faciliation is blocked by CaM1234 Calmodulin is tethered to the L-type Ca 2+ channel

  15. Lobe specific regulation by Calmodulin Wt CaM enhances CDI CaM1234 inhibits CDI CaM12 enhances CDI CaM34 inhibits CDI

  16. Possible models for CDI of L-type Ca2+ channels Pitt et al., 2001 Active site remodeling Erickson et al., 2003

  17. Facilitated open Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ Channels C C C C N N C N C C Normal open Normal inactivated CaM facilitates opening CaM enhances CDI Facilitated inactivated C Resting

  18. CaM mediates R-and N-Type channel CDI, via the interaction of the N-lobe CaM R-Type channel L-Type channel

  19. C N C N N N C C L-Type Not found CDI P/Q-Type CDI CDF Not found N-Type CDI R-Type CDI Not found Buffer sensitive Buffer insensitive Detects global Ca 2+ entry Detects local Ca 2+ entry (N-lobe) modulated (C-lobe) modulated C N slow VDI Fast VDI EF + IQ preIQ

  20. CaM LGCC VGCC SOCC MSCC R NCX Second messenger NCX Mitochondria PMCA IP3R RYR IP3R SPCA1 Others?? Uniporter Golgi ER/SR SERCA

  21. 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

  22. C N Lobe dependent regulation of RyR1 by calmodulin Ca2+ free CaM functions as an agonist C2 C1 N2 N1

  23. Lobe dependent regulation of RyR1 by calmodulin Ca2+ free CaM functions as an antagonist C2 C1 C N N2 N1

  24. C N Lobe dependent regulation of RyR1 by calmodulin Ca2+ free CaM functions as an agonist C2 C1 N2 N1

  25. Lobe dependent regulation of RyR1 by calmodulin C-lobe Movement driven by Ca2+ Ca2+ C2 C1 C N N2 N1

  26. Lobe dependent regulation of RyR1 by calmodulin Ca2+ CaM functions as an inhibitor C2 C1 C N N2 N1

  27. C N Lobe dependent regulation of RyR1 by calmodulin Ca2+ free Agonist C2 C1 C2 C1 C N N2 N1 N2 N1 Ca2+ Ca2+ Inhibitor C2 C1 C2 C1 C C N N N2 N1 N2 N1

  28. Binding of apocaM and Ca2+CaM to the RyR1

  29. NMDA receptors: Lobe dependent interaction of CaM

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

  31. Ca2+ permeant channels K+ channels PMCA CaM CaM RyR CaM CaM Low Ca 2+

  32. Ca2+ permeant channels K+ channels PMCA CaM CaM CaM RyR CaM High Ca 2+

  33. Ca2+ permeant channels K+ channels PMCA CaM CaM CaM RyR CaM High Ca 2+ Calmodulin: Mediator of the calcium Modulation of Multiple Ion Channels

  34. Calcium-Binding Protiens: Intracellular sensors from the Calmodulin Superfamily

  35. CaM CaM EF1 EF2 EF3 EF4 CaBP1-5 EF1 EF2 EF3 EF4 CaBP7-8 EF1 EF2 EF3 EF4 EF3 EF4 GCAPs EF1 EF2 NCS1 EF1 EF2 EF3 EF4 CaM-like L-CaBP1 S-CaBP1 Caldendrin S-CaBP5 S-CaBP2 L-CaBP2 S-CaBP1 CaBP3 GCAP GCAP3 GCAP2 Recoverin Recoverin Visinin EF1 EF2 EF3 EF4 Neurocalcin VILIP3 VILIP1 VILIP2 NCS1 Adapted from Haeseleer et al., 2000

  36. Basal Ca2+ 100 80 NCS1 VILIP-1 CaM Synaptotagmin 60 Ca2+ bound 40 20 0 7 6 5 4 3 -log[Ca2]+ Why so many CaBPs….? Cellular Localisation and…. Exocytosis in Synapses Exocytosis in Endocrine cells Channel regulation Adapted from Burgoyne and Weiss 2001

  37. CaBP: intracellular sensors for the calmodulin superfamily Extra helical turn Adapted from Haeseleer et al., 2002

  38. CaBPs are myristoylated and targeted to the membrane GFP control sCaBP1 lCaBP1 Haeseleer et al. 2000

  39. Recoverin: member of the Neuronal calcium sensors Ca 2+ -induced myristoyl switch

  40. 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 Only inhibitory action Ca2+-independent NCS1 was shown to mediate a rapid ca2+ dependent Facilitation P/Q -type Ca2+ channel and enhances inactivation Ca2+ independent Why different: different affinities for Ca2+ Calmodulin and CaBP1 are direcly bound to the channel

  41. New type of Ca2+ -induced Ca2+ release (CICR) mechanisme in A7r5 cells 100 50 0 10 20 0 45Ca2+ -flux on permeabilized A7r5 cells Ionophore Permeabilized 45Ca2+ Loaded Intact IP3 Ca2+ ER ER ER Fractional loss (%/2 min)

  42. Ca2+ dependence: EC50 = 700 nM Hill = 1.9 Mg2+ inhibition: EC50= 0.6 mM ATP stimulation: EC50= 320 µM Characteristics of the CICR mode

  43. 40 30 20 Fractional loss (%/ 2 min) 10 0 0 10 20 Time (min) control CaM1234 CaM Effects of CaM and CaM1234 on CICR

  44. CaM EF1 EF2 EF3 EF4 CaBP1 short/long EF1 EF2 EF3 EF4 NCS1 NCS1 3 EF1 EF1 EF2 EF2 EF3 EF3 EF4 EF4

  45. Preincubation with a CaM-binding peptide inhibits CICR control RyR1 CaM-BS (peptide aa 3614-3643) Ca2+

  46. CaM but not CaM1234 can restore CICR CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) Ca2+

  47. CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) CaM Ca2+

  48. CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) CaM1234 CaM Ca2+

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