1. Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -binding Proteins

2. Introduction: Calcium signalling Inositol trisphosphate receptor (IP3R) Results: Regulation of the IP3R by Ca2+ and calmodulin Regulation of intracellular Ca2+ release by neuronal Ca2+ binding proteins A novel Ca2+-induced Ca2+ release mechanism in A7r5 and 16HBE014-cells Conclusions Perspectives

3. Ca2+

4. VGCC LGCC SOCC (TRP) MSCC PMCA NCX RYR ER/SR SERCA IP 3 R Golgi PMR1 Mitochondria NCX Uniporter Buffers R Second messenger IP 3 R Others??

5. Introduction: Calcium signalling Inositol trisphosphate receptor (IP3R) Results: Conclusions Perspectives Regulation of the IP3R by Ca2+ and calmodulin Regulation of intracellular Ca2+ release by neuronal Ca2+ binding proteins A novel Ca2+-induced Ca2+ release mechanism in A7r5 and 16HBE014-cells

7. Introduction IP 3 R Regulation of the IP 3 R by Ca 2+ and CaM Results Localization and characterization of the CaM-binding sites Function of CaM Regulation of the IP 3 R by Neuronal Ca 2+ -binding proteins Conclusions

8. IP 3 R I, II, III Agonists IP 3 Ca 2+ Intraluminal proteins: Chromogranins; Calnexin, Calreticulin Cytosolic proteins: Calmodulin; CaBP IRBIT; CARP Cytoskeletal proteins: Actin; MyosinII Ankyrin; Talin; Vinculin 4.1N Plasma membrane associated: Homer-mGluR TRPs; G  Kinases and phosphatases: PKA; PKC BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1  ER cytosol

9. Ca 2+ is the primary modulator of its own release Structure of the IP 3 R Regulation of the IP 3 R by Ca 2+ Bell-shaped IICR Hamada et al., 2002 Bezprozvanny et al., 1991

10. IICR Regulation of the IP 3 R by CaM + CaM (Missiaen et al., 1999) [Ca 2+ ] (nM)300 - CaM (Michikawa et al., 1999) CaM Ca 2+ dependently inhibits IP 3 R I, II and III IP 3 R I II III

11. Calmodulin binding sites on IP 3 R1 13 Endoplasmic reticulum Cytosol R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA CaCaM CaM (Adkins et al,2000) W1577A (Zhang et al, 2001; Nosyreva et al, 2002)

12. Localization and characterization of the CaM-binding sites Results

13. Calmodulin effects on IP 3 binding CaM inhibits IP 3 binding in Ca 2+ -independent way C N 0510 0.0 0.1 0.2 0.3 B/F Bound (nM) [ 3 H]IP 3 binding (%) Control CaM Ca 2+ /CaM CaM 1234 Ca 2+ /CaM 1234 Ca 2+ IP 3 binding core 581 226581 1-225 1

14. Localisation of a calmodulin-binding site GST-fusion protein pull down of CaM 1234 CaM 1234 pGST GST-Cyt1 GST-Cyt2 50  M free Ca 2+ 1 mM EGTA CaM 1234 Cyt1 Cyt2 1 159 309 IP 3 binding core 581 226581 1-225 1

15. Detailed localisation using peptides A B C D E F CaM A B C E D F 1 159 1-5-10 1-5-8-14 70% IQ 76% IQ 53% IQ A B C D E F CaM Ca 2+ EGTA Discontinue Ca 2+ -independent CaM-binding site in the N- terminal region

16. A B C E D F 1159 control∆ B∆ E [ 3 H]IP 3 binding Both CaM-binding sites are essential for inhibiting IP 3 binding CaM-+ + + - -

17. Calmodulin-binding sites on IP 3 R1 13 ER Cytosol R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD CaCaM CaMCa 2+

18. 0.5 µM1 µM100 µM Control CaM IICR is inhibited by CaM and CaM 1234 Time (s) ATP 600 400 200 Ca 2+ i (nM) 1000750500250 0 0 CaM 1234 CaM CaM 1234 Control

19.  CaM is not the Ca 2+ sensor for the IP 3 R 200 nM IP 3 CaM 1234 CaM [Ca 2+ ] (µM) 45 Ca 2+ flux 0.11 IICR is inhibited by CaM and CaM 1234 50 0 Ca 2+ release (%)

20. Half CaM’s CaM C-CaM N-CaM 3 HIP 3 binding control 45 Ca 2+ flux control Both CaM lobes are necessary to inhibit the IP 3 R

21. Ca 2+ Closed « square » Open « windmill » IICR [Ca 2+ ] (nM)300 Inactive Other regulators kinases phosphatases The N-terminal Ca 2+ -independent CaM-binding Site on the IP 3 R is Responsible for CaM Inhibition, even though this Inhibition Requires Ca 2+

22. IP 3 R1IP 3 R3 suramin

23. Summary  Two CaM-binding sites on the IP 3 R Ca 2+ dependent in the regulatory domain Ca 2+ independent in the N-terminus  CaM inhibits IICR only in the presence of Ca 2+  CaM inhibits IICR by lowering the affinity for IP 3, CaM is not the Ca 2+ sensor

24. Introduction: Calcium signalling Inositol trisphosphate receptor (IP3R) Results: Conclusions Perspectives Regulation of the IP3R by Ca2+ and calmodulin Regulation of intracellular Ca2+ release by neuronal Ca2+ binding proteins A novel Ca2+-induced Ca2+ release mechanism in A7r5 and 16HBE014-cells

25. Regulation of calcium release by neuronal Calcium Binding Proteins (CaBP)

26. CaM CaM-like L-CaBP1 S-CaBP1 Caldendrin S-CaBP5 S-CaBP2 L-CaBP2 S-CaBP3 CaBP3 GCAP GCAP3 GCAP2 Recoverin Visinin Neurocalcin VILIP3 VILIP1 VILIP2 NCS1 EF1EF2EF3EF4 CaM EF1EF2EF3EF4 CaBP1 EF1EF2EF3EF4 CaBP2 EF3EF4 CaBP3 EF1EF2EF3EF4 CaBP4 EF1EF2EF3EF4 CaBP5 Adapted from Haeseleer et al., 2000 CaBP NCS

27. Why so many CaBPs….? Cellular localisation Ca 2+ myristyol switch Burgoyne et al., 2004 NUCLEUS Golgi Low Ca 2+ NUCLEUS High Ca 2+ Golgi * * * * *

28. Exocytosis in Endocrine cells Exocytosis in Synapses Channel regulation Basal Ca2+ NCS1VILIP-1CaMSynaptotagmin 7 6543 100 80 60 40 20 0 Ca 2+ bound -log[Ca 2 ] + Adapted from Burgoyne and Weiss 2001 Why so many CaBPs….? Cellular Localisation and….

29. CaBPs agonist of the IP 3 R? Taken from Yang et al., 2002 CaBPs are agonists of the IP 3 R CaBPs bind to 1-600 N-terminus of the IP 3 R CaBPs binding is Ca 2+ dependent CaBPs co-localize with the IP 3 R

30. sCaBP1 GST 1-604 GST 1-225 GST 226-604 CaBP1 binds to the IP 3 R NH 2 COOH ER CYT IP 3 binding core (226- 581) caldendrin lCaBP1 sCaBP1

31. A B C E D F 1 159 CaM sCaBP1 ABCDEF ABCDEF + Ca 2+ EGTA CaBP1 binds to a similar region of the IP 3 R as CaM

32. CaBP 1/11/21/41/5 1/6 1/8 1/10 1/3 CaBP Ratio of CaBP: peptide B 0246810 0.0 0.5 1.0 Band intensity Peptide B: CaBP + Calcium + EGTA Binding of CaBP to the IP 3 R is Ca 2+ independent

33. EF1EF2EF3EF4 CaBP1 lCaBP1 sCaBP1 Cellular localization of CaBP1

34. sCaBP 0.5µM 1µM 100µM ATP Control lCaBP 02004006008001000 0 Time (s) 1000 200 400 600 800 Ca 2+ i (nM) Both Long and Short CaBP1 inhibit IICR

35. SCaBP1 (n=22) LCaBP1 (n=17) Control (n=44) * * * *

36. CaBP overexpression inhibits IP 3 Ester induced Calcium release 200 150 100 120010008006004002000 10  M InsP 3 ester 0 50 250 SCaBP1 Control time (s) Ca2+ i (nM) CaBP acts directly on the IP 3 R

37. CaBP1 inhibits IP 3 binding to the IP 3 R control sCaBP1Ca 2+ sCaBP1 100 [ 3 H]IP 3 Binding (% vs control) 75 50 0 25 IP 3 binding 45 Ca 2+ flux 45 Ca 2+ -release (%) [IP 3 ] (µM) control sCaBP1 1 10 1000,1 70 50 30 10 90 CaBP acts directly on the IP 3 R by inhibiting IP 3 binding

38. EF1EF2EF3EF4 1000 800 600 400 200 01500 5001000 Time (s) 0 Ca 2+ i (nM) 0.51100 µM ATP Control CaBP 134 CaBP1 inhibits IICR independent of Ca 2+ binding (1) 100 50 0 % responsive cells 0.51100 ATP (  M)

39.  CaBP1 is not a Ca 2+ sensor for the IP 3 R 200 nM IP 3 CaBP1 134 CaBP1 [Ca 2+ ] (µM) 45 Ca 2+ flux (%) 0.11 50 0 CaBP1 inhibits IICR independent of Ca 2+ binding (2)

40. IICR is inhibited by CaBP1 CaBP1 binds to the receptor in a Ca 2+ independent manner resulting in inhibition of IP 3 binding to its receptor. To summarise.. But, how is CaBP1 activity regulated? CaBP does not activate Ca 2+ release as previously reported

41. sCaBP1sCaBP1-G2A lCaBP1-G2AlCaBP1 Membrane Targeting of CaBP may regulate its activity (1) EF1EF2EF3EF4 CaBP1-G2A

42. ATP (µM ) Membrane Targeting of CaBP1 may regulate its activity (2) 0 1500 500 1000 time (s) 500 750 1000 0.5 1 100 Control 250 0 sCaBP -G2A Ca 2+ i (nM) 900 100 1 0.5 100 50 0 % responsive cells 700 500 300 100 0 ATP (  M) Peak amplitude (nM) * * * *

43. 32 P- CaBP YFPS120A wt 0.51 01500 500 1000 Time (s) 0 400 800 1200 Ca 2+ i (nM) ATP (µM) 100 Control CaBP1 S120A 1000 100 1 0.5 500 0 ATP (  M) Peak amplitude (nM) * * 100 50 0 % responsive cells * * * Phosphorylation of CaBP1 decreases it’s potency EF1EF2EF3EF4 S120A CaBP1 S120A Casein kinase [ST]xx[DE]

44. 10µM CCh 0.5mM Caf 1mM Caf 2mM Caf 0 100 200 300 [ C a 2 + ] i ( n M ) CaBP1 does not Affect Ca 2+ release through RyRs 5000100015002000 Time (s) Control sCaBP1

45. Summary  CaBP inhibits IICR  CaBP binds to the IP 3 R inhibiting IP 3 binding  CaBP activity is Ca 2+ independent  Myristoylation is not essential for CaBP activity, although it may have a regulatory role  Phosphorylation of CaBP decreases it’s potency  RyRs are not modulated by CaBP

46. CaBP1CaM Neurons IP 3 RRyR In Neurons: CaM preferentially modulates RyRs CaBPs preferentially modulate IP 3 R? Conclusions

47. The End

48. VGCC LGCC SOCC (TRP) MSCC PMCA NCX IP 3 RRYR ER/SR SERCA IP 3 R Golgi PMR1 Mitochondria NCX Uniporter Others?? Buffers Second messenger

49. Control Calbindin 0.5µM ATP1µM ATP100µM ATP 0 200 400 600 800 0 1500 5001000 Time (s) Ca2+ i (nM) Inhibition of IICR by CaBP is not due to Calcium Buffering

50. % responsive cells 100 50 0 100 200 300 400 500 100  M1M1M0.5  M 0 Peak Amplitude (nM) ATP Control Calbindin Calbindin Decreases Peak Response without decreasing number of responsive cells

51. Introduction: Calcium signalling Inositol trisphosphate receptor (IP3R) Results: Conclusions Perspectives Regulation of the IP3R by Ca2+ and calmodulin Regulation of intracellular Ca2+ release by neuronal Ca2+ binding proteins A novel Ca2+-induced Ca2+ release mechanism in A7r5 and 16HBE014-cells

52. Introduction: Calcium signalling Inositol trisphosphate receptor (IP3R) Results: Conclusions Perspectives Regulation of the IP3R by Ca2+ and calmodulin Regulation of intracellular Ca2+ release by neuronal Ca2+ binding proteins A novel Ca2+-induced Ca2+ release mechanism in A7r5 and 16HBE014-cells