1. Regulation of intracellular Ca 2+ -release channels by Ca 2+ and Ca 2+ -binding proteins Nael Nadif Kasri September 21st, 2004

2. 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP 3 R) 3. Results 4. Conclusions and future perspectives Regulation of the IP 3 R by Ca 2+ and calmodulin Regulation of intracellular Ca 2+ release by neuronal Ca 2+ -binding proteins A novel Ca 2+ -induced Ca 2+ release (CICR) mechanism in A7r5 and 16HBE014-cells Overview 2. Aims

3. Death Calcium: a universal second messenger! life brain muscle heart

4. Ca 2+ channels RyR ER/SR SERCA IP 3 R Golgi SPCA1 Mitochondria Ca 2+ buffers/sensors PMCA NCX IP 3 R Intracellular calcium homeostasis PTP Uniporter [Ca 2+ ]~ 1 mM [Ca 2+ ]~ 100 nM

5. GPCR γ β α PLC PKC IP 3 R activation PIP 2 DAG IP 3 ER Cytosol

6. Structure of the IP 3 R 13 Endoplasmic reticulum Cytosol Agonists IP 3 and Ca 2+ Ca 2+ sensor CaM ATP P P P P NH 2 COOH Tetramer 3 isoforms (I, II, III) IP 3

7. 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 Windmill Square

8. Aims Regulation of the Ca 2+ dependent bell-shaped activation curve Calmodulin Neuronal Ca 2+ -binding proteins

9. 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP 3 R) 3. Results 4. Conclusions and future perspectives Regulation of the IP 3 R by Ca 2+ and calmodulin Regulation of intracellular Ca 2+ release by neuronal Ca 2+ -binding proteins A novel Ca 2+ -induced Ca 2+ release (CICR) mechanism in A7r5 and 16HBE014-cells Overview 2. Aims

10. Calmodulin CaM: Ca 2+ -sensor protein 4 EF hands closedopen apoCaMCa 2+ CaM 2 conformations: EF1EF2EF3EF4

11. IICR Regulation of the IP 3 Rs by CaM + CaM (Missiaen et al., 1999) [Ca 2+ ] (nM) 300 -CaM (Michikawa et al., 1999) IP 3 R I II III Ca 2+ -dependentIP 3 - dependent Inhibition of IICR IP 3 R I II III [IP 3 ] (µM) + CaM

12. 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)

13. Calmodulin effects on IP 3 binding CaM inhibits IP 3 binding in a Ca 2+ -independent way C N IP 3 binding core 581 IP 3 binding core 226581 suppressor 1 Control [ 3 H]IP 3 binding (%) 100 60 40 20 80 CaM 1234 Ca 2+ /CaM 1234 Ca 2+ /CaM Ca 2+ CaM 0510 0.0 0.1 0.2 0.3 B/F Bound (nM) + CaM

14. 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

15. control ∆ B∆ E [ 3 H]IP 3 binding (%) vs control Both CaM-binding sites are essential for inhibiting IP 3 binding CaM-+ + + - - IP 3 binding core 581 suppressor 1 BE Both sites essential? 0 20 40 60 80 100

16. 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 Ca 2+ -indep CaM Ca 2+ CaM effects are Ca 2+ -dependent! CaM 1234 ?

17. 0.5 µM1 µM100 µM Control CaM IICR is inhibited by CaM and CaM 1234 (1) Time (s) ATP 600 400 200 Ca 2+ i (nM) 1000750500250 0 0 CaM 1234 Intact COS cells Decreased amount of responsive cells Increased latency

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

19. Role of half CaM’s CaM Both CaM lobes are necessary to inhibit the IP 3 R C-CaM N-CaM Control 45 Ca 2+ release vs A23187 (%) 100 60 40 20 80 45 Ca 2+ flux Control [ 3 H]IP 3 binding vs control (%) 100 60 40 20 80 [ 3 H]IP 3 binding

20. Ca 2+ Closed « square » Open « windmill » IICR [Ca 2+ ] (nM)300 Inactive Other regulators Ca 2+ kinases phosphatases To summarise…

21. 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP 3 R) 3. Results 4. Conclusions and future perspectives Regulation of the IP 3 R by Ca 2+ and calmodulin Regulation of intracellular Ca 2+ release by neuronal Ca 2+ -binding proteins A novel Ca 2+ -induced Ca 2+ release (CICR) mechanism in A7r5 and 16HBE014-cells Overview 2. Aims

22. Family of neuronal Ca 2+ -binding proteins EF-hand containing Ca 2+ sensors, CaM-like proteins Expressed in brain and retina N-terminal Myristoylation: subcellular targetting to plasmamembrane and TGN One or more EF-hands are unable to bind Ca 2+ EF1EF2EF3EF4 CaBP1 CaM EF1EF2EF3EF4 Recoverin EF1EF2EF3EF4

23. sCaBP1 GST 1-604 GST 1-225 (CaM-binding site) GST 226-604 CaBP1 binds to the IP 3 R caldendrin lCaBP1 sCaBP1 EF1EF2EF3EF4 CaBP1 Novel agonist of the IP 3 R?? (Yang et al., 2002)

24. 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 Binding of CaBP1 to the IP 3 R is Ca 2+ -independent

25. sCaBP1 0.5µM 1µM 100µM ATP Control lCaBP1 02004006008001000 0 Time (s) 1000 200 400 600 800 Ca 2+ i (nM) Both Long and Short CaBP1 inhibit IICR Decreased amount of responsive cells Increased latency Intact COS cells

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

27. EF1EF2EF3EF4 YFP 1000 800 600 400 200 01500 5001000 Time (s) 0 Ca 2+ i (nM) 0.51100 µM ATP Control CaBP1 134 CaBP1 inhibits IICR independent of Ca 2+ binding CaBP1 activity is regulated by: Myristoylation Phosphorylation

28. 0 100 200 300 [ C a 2 + ] i ( n M ) CaBP1 does not affect Ca 2+ release through RyRs 5000100015002000 Time (s) Control sCaBP1 10µM CCh 0.5mM Caf 1mM Caf 2mM Caf RyRs are not modulated by CaBPs: In neurons: CaBP1 CaM IP 3 RRyR To summarise…

29. 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP 3 R) 3. Results 4. Conclusions and future perspectives Regulation of the IP 3 R by Ca 2+ and calmodulin Regulation of intracellular Ca 2+ release by neuronal Ca 2+ -binding proteins A novel Ca 2+ -induced Ca 2+ release (CICR) mechanism in A7r5 and 16HBE014-cells OVERVIEW 2. Aims

30. New type of Ca 2+ -induced Ca 2+ release (CICR) 45 Ca 2+ -flux on permeabilized A7r5 and 16HBE14o-cells ER Intact Permeabilized 45 Ca 2+ Loaded ER IP 3 Ca 2+ CICR is not mediated by IP 3 R nor RyR EC 50 : 700 nM 0 20 10 0 50 100 Fractional loss (%/2 min) Time (min)

31. Effects of CaM and CaM 1234 on CICR Fractional loss (%/ 2 min) Time (min) 01020 10 20 30 40 CaM 1234 control CaM CICR is inhibited by: CaM 1234 CaM 1 CaBP1 NCS1 Ca 2+ CaM Ca 2+ sensor?

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

33. Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) Ca 2+ CaM but not CaM 1234 can restore CICR

34. Ca 2+ CaM Preincubation with RyR1 CaM-BS (peptide aa 3614-3643)

35. Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) Ca 2+ CaM CaM 1234 CaM but not CaM 1234 can restore CICR

36. New type of Ca 2+ -induced Ca 2+ release channel ?? IP 3 RCICR CaM To summarise… 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)? CICR channel Ca 2+ + ATP + CaM is the Ca 2+ sensor Mg 2+ - CaM 1234 - Inhibited by CaM mutants Inhibited by CaM-like proteins

37. Conclusions CICR ER IP 3 R RyR CaM CaBP1

38. Future Perspectives   Role of neuronal Ca 2+ -binding proteins in neurons   Identification of CICR channel

39. Thank You