1. IP 3 -induced Ca 2+ release and calmodulin Laboratory of Physiology KULeuven Leuven, Belgium

2. IP 3 R I, II, III Agonists IP 3 Ca 2+ Intraluminal proteins: Chromogranins; Calnexin Cytosolic proteins: Calmodulin; CaBP IRBIT CARP HAP1A-Htt Cytoskeletal proteins: Actin; MyosinII Ankyrin; Tallin; Vinculin 4.1N Plasma membrane associated: Homer-mGluR TRPs; RhoA-TRPC1 G  Kinases and phosphatases: PKA; Fyn BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1 

3. Calmodulin inhibits IP 3 -induced Ca 2+ release A7r5HBE A7r5 cells 70% IP 3 R1 CaM Control HBE cells 90% IP 3 R3 Control CaM

4. Lbs-1: IP 3 - binding core (226-581) aa 1-225 NH 2 COOH ER CYT Lbs-1 Lbs-1  1-225 1 581 W226581 1-225 Recombinant ligand-binding domain of IP 3 R1 (LBS-1) CaM at N-terminal site inhibits IP 3 binding CaM Adkins et. al., 2000 Ca 2+ CaM Yamada et. al., 1995 Lin et. al., 2000 Ca 2+ CaM SII

5. Ca 2+ CaM1234 10 µM CaM1234 Ca 2+ CaM 10 µM apoCaM 5 µM Ca 2+ contr ol [ 3 H]IP 3 binding (%) Lbs-1His Lbs-1  1-225His 1 581 W226581 0510 0.0 0.1 0.2 0.3 B/F Bound (nM ) EC 50 = 1.7µM The inhibition is Ca 2+ independent

6. A B C E D F 1 159 1-5-10 1-5-8-14 70% IQ (site1) 76% IQ 53% IQ CaM-binding sites in the N-terminal region are Ca 2+ independent Ca 2+ independent EC 50  1-1.5 µM A B C D E F CaM Ca 2+ A B C D E F CaM EGTA

7. 1000750500250 0 600 400 200 0 0.51100 ATP (µM ) Ca 2+ i (nM) Control CaM In COS cells IICR is inhibited by CaM and by CaM1234 CaM1234

8. 40 Ca 2+ release vs A23187 (%) 50 60 70 80 90 100 0 1 2 3 4 5 control CaM CaM1234 In permeabilized L15 cells IICR is inhibited by CaM and by CaM1234 L15 cells IP 3 R1IP 3 R3 CaM is not the Ca 2+ sensor for the inhibition of IICR [Ca 2+ ] µM %

9. The Ca 2+ /CaM site in the coupling region is not involved in the CaM inhibition of IICR R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD Ca 2+ /CaM W1577A (Zhang et al, 2001; Nosyreva et al, 2002)

10. CONCLUSION: The N-terminal Ca 2+ -independent CaM-binding site is responsible for the CaM inhibition of IICR 1) Interaction site for other CaM-like proteins ? 2) Involved in intramolecular interactions? Significance ?

11. Adapted from Haeseleer et al., 2000 CaM or other CaM-like Ca 2+ sensor proteins ? Interaction with neuronal Calcium Binding Proteins (CaBP) Inhibitory Activatory ? (Yang et al., 2002)

12. CaBP1 GST 1-581 GST 1-225 GST 226-581 CaBP1 binds to the N-terminal part of the IP 3 R CaBP1 ? NH 2 COOH ER CYT IP 3 binding core (226-581) aa 1-225 Ca 2+ CaM CaM

13. A B C E D F 1 159 CaM sCaBP1 ABCD E F ABCDEF + Ca 2+ -Ca 2+ / EGTA A) B) CaBP1 binds to a similar region as CaM independently of Ca 2+

14. Control 02004006008001000 200 400 600 800 0 sCaBP lCaBP 0.5µM 1µM 100µM ATP Time (s) Ca 2+ i (nM) Both long and short CaBPs inhibit IP 3 -induced calcium release in COS-1 cells 45 Ca 2+ flux

15. CaBP inhibits IP 3 -induced Ca 2+ release independent of Ca 2+ binding EF1EF2EF3EF4 CaBP134 1000 800 600 400 200 01500 5001000 Time (s) 0 [Ca 2+ ] i (nM) 0.51100 µM ATP Control YFP

16. Significance ? 1) Interaction site for other CaM-like proteins ? 2) Involved in intramolecular interactions?

17. Suramin interacts with the CaM-binding sites on IP 3 R1 1-225 EGTA Ca 2+ IP 3 R1 Input Seph CaM-Seph + Suramin Seph CaM-Seph + Suramin EGTA Ca 2+ Control 10 µM CaM Control 100 µM suramin Suramin mimics the CaM inhibition of IICR

18. IP 3 binding core (IBC) NH 2 COOH ER CYT 1-->225 CaM Ca 2+ CaM Interaction 1-225 with IBC GST / IP 3 AdPhosCaM sCaBP-1 N-terminal CaM-binding site = intramolecular interaction site? SuraminCABP1 CaM1234

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

20. New type of Ca 2+ -induced Ca 2+ release (CICR) channel ?? + CaM1234

21. 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

22. 01020 0 10 20 30 40 Fractional loss (%/ 2 min) Time (min) Effects of CaM, CaM 1 and CaM 1234 control CaM CaM 1 CaM 1234

23. CalmodulinCalmodulin1234 Long CaBP1 NCS-1/Frequenin Short CaBP1 NCS-1/FrequeninE120Q Calmodulin1 Calmodulin Calmodulin1234 Long CaBP1 NCS-1/Frequenin Short CaBP1 NCS-1/FrequeninE120Q Calmodulin1234 Long CaBP1 NCS-1/FrequeninNCS-1/Frequenin Short CaBP1 NCS-1/FrequeninE120Q Calmodulin1

24. Ca 2+ (3 µM) control RyR1 CaM-BS (peptide aa 3614-3643) Preincubation with a CaM-binding peptide inhibits CICR

25. CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) Ca 2+ (3 µM) CaM

26. CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) Ca 2+ (3 µM) CaM1234 CaM

27. 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+ - CaM1234 - Inhibited by CaM mutants Inhibited by CaM-like proteins

28. Jan B. PARYS Geert CALLEWAERT Ludwig MISSIAEN Rafael A. FISSORE Nael NADIF KASRI Geert BULTYNCK Karolina SZLUFCIK Leen VERBERT Elke VERMASSEN Zerihun ASSEFA Iris CARTON Patrick DE SMET Babraham Institute Cambridge UK H. Llewelyn Roderick Martin D. Bootman Michael Berridge KULeuven Leuven, Belgium Andreas Jeromin Baylor College of Medicine Houston, Texas The Division of Molecular Neurobiology The Institute of Medical Science The University of Tokyo Katsuhiko Mikoshiba