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

2. 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

3. 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; Fyn BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1  ER cytosol

4. 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

5. 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

6. 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)

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

8. 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

9. 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

10. 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

11. 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-+ + + - -

12. 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+

13. 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

14.  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 (%)

15. 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

16. Ca 2+ closed open IICR [Ca 2+ ] (nM) 300 inactive Other regulators PKC? 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+

17. 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

18. 1-225 EGTACa 2+ IP 3 R1 input - Seph CaM-Seph Seph Suramin +-+- Suramin interacts with the CaM-binding sites on the IP 3 R1 (1) Suramin: polysulfonated naphthylurea Interacted with the RYR CaM-binding sites (papineni et al., 2002)

19. Suramin interacts in a direct way with the CaM-binding sites Suramin CaM BE 1 159 B E 0 µM100 µM0 µM100 µM Suramin interacts with the CaM-binding sites on the IP 3 R1 (2)

20. Suramin inhibits IICR Does Suramin counteract the effects of CaM? IP 3 R1IP 3 R3 suramin Contr ol 10 µM CaM Missiaen et al., 1999 Suramin inhibits IICR

21. IP 3 binding core 581 226581 1-225 1 Suramin + - +- [ 3 H]IP 3 Binding Suramin inhibits IP 3 binding [ 3 H]IP 3 Binding [Suramin] (µM) B/F Bound (nM) 05 suramin

22. Summary  Suramin inhibits IICR by lowering the IC 50 for activation by IP 3  Suramin interacts with the CaM-binding sites on the IP 3 R Suramin is a potent inhibitor of IICR Suramin mimicks the effects of CaM

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

24. 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

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

26. 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….

27. 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

28. 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

29. 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

30. 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

31. EF1EF2EF3EF4 CaBP1 lCaBP1 sCaBP1 Cellular localization of CaBP1

32. 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

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

34. 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

35. 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

36. 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)

37.  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)

38. 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

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

40. 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) * * * *

41. 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]

42. 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

43. Summary  CaBP inhibits IICR  CaBP binds to the InsP 3 R inhibiting IP 3 binding  CaBP activity is Calcium independent  Myristoylation is not necessary for CaBP activity, although it may have a regulatory role  Phosphorylation of CaBP decreases it’s potency  RyRs are not modulated by CaBP In Neurons: CaM preferentially modulates RyRs CaBPs preferentially modulate IP 3 R?

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

45. 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

46. 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

47. % 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