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Regulation of intracellular Ca 2+ -release channels by Ca 2+ and Ca 2+ -binding proteins

Regulation of intracellular Ca 2+ -release channels by Ca 2+ and Ca 2+ -binding proteins. Nael Nadif Kasri. September 21st, 2004. Overview. 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP 3 R). 2. Aims. 3. Results.

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Regulation of intracellular Ca 2+ -release channels by Ca 2+ and Ca 2+ -binding proteins

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  1. Regulation of intracellular Ca2+-release channels by Ca2+ and Ca2+-binding proteins Nael Nadif Kasri September 21st, 2004

  2. Overview 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP3R) 2. Aims 3. 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 (CICR) mechanism in A7r5 and 16HBE014-cells 4. Conclusionsand future perspectives

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

  4. Intracellular calcium homeostasis Ca2+ channels NCX IP3R RyR PMCA IP3R SPCA1 SERCA Uniporter Ca2+ buffers/sensors [Ca2+ ]~ 1 mM [Ca2+ ]~ 100 nM PTP Mitochondria ER/SR Golgi

  5. γ β α IP3R activation GPCR DAG PIP2 PLC PKC IP3 ER Cytosol

  6. Structure of the IP3R ATP P CaM P P ATP 31 25 ATP Ca2+ sensor P 18 Cytosol Tetramer NH2 3 isoforms (I, II, III) IP3 Agonists IP3and Ca2+ COOH 13 Endoplasmic reticulum

  7. Regulation of the IP3R by Ca2+ Bell-shaped IICR Structure of the IP3R Bezprozvanny et al., 1991 Hamada et al., 2002 Square Windmill Ca2+ is the primary modulator of its own release

  8. Aims Regulation of the Ca2+ dependent bell-shaped activation curve Calmodulin Neuronal Ca2+ -binding proteins

  9. Overview 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP3R) 2. Aims 3. 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 (CICR) mechanism in A7r5 and 16HBE014-cells 4. Conclusions and future perspectives

  10. EF1 EF2 EF3 EF4 Calmodulin CaM: Ca2+-sensor protein 4 EF hands 2 conformations: apoCaM Ca2+CaM closed open

  11. IP3R I II III + CaM [IP3] (µM) Regulation of the IP3Rs by CaM IICR • CaM • (Michikawa et al., 1999) IP3R I II III + CaM (Missiaen et al., 1999) 300 [Ca2+] (nM) Inhibition of IICR Ca2+-dependent IP3- dependent

  12. Calmodulin-binding sites on IP3R1 W1577A (Zhang et al, 2001; Nosyreva et al, 2002) R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA 31 25 18 Cytosol CaM?? (Adkins et al,2000) CaCaM 13 Endoplasmic reticulum

  13. Calmodulin effects on IP3 binding N 226 581 C IP3 binding core 100 80 [3H]IP3 binding (%) 60 40 + CaM 0.3 20 0.2 B/F 0.1 Control Ca2+ CaM CaM1234 Ca2+/CaM1234 Ca2+/CaM 0.0 0 5 10 Bound (nM) 1 581 IP3 binding core suppressor CaM inhibits IP3 binding in a Ca2+ -independent way

  14. Detailed localisation using peptides 159 1 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ A C D E B F A B C D E F CaM Ca2+ EGTA A B C D E F CaM Discontinue Ca2+-independent CaM-binding site in the N-terminal region

  15. 1 581 IP3 binding core suppressor Both sites essential? B E ∆ B ∆ E control 100 80 60 [3H]IP3 binding (%) vs control 40 20 0 CaM + + - - - + Both CaM-binding sites are essential for inhibiting IP3 binding

  16. Calmodulin-binding sites on IP3R1 CaM1234? 31 25 18 Ca2+ -indep CaM Cytosol R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD Ca2+ CaM effects are Ca2+-dependent! R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA CaCaM 13 ER

  17. 600 CaM 400 Ca2+i (nM) 200 CaM1234 0 0 250 500 750 1000 IICR is inhibited by CaM and CaM1234 (1) Intact COS cells 0.5 µM 1 µM 100 µM ATP Control Time (s) Decreased amount of responsive cells Increased latency

  18. 200 nM IP3 IICR is inhibited by CaM and CaM1234 (2) 45Ca2+ flux Permeabilized cells (%) 200 nM IP3 CaM 50 Ca2+ release 200 nM IP3 CaM1234 0 0.1 1 [Ca2+ ] (µM) • CaM is not the Ca2+ sensor for the IP3R

  19. Role of half CaM’s N-CaM C-CaM [3H]IP3 binding 45Ca2+ flux 100 100 80 80 60 60 45Ca2+ release vs A23187 (%) [3H]IP3 binding vs control (%) 40 40 20 20 Control Control CaM Both CaM lobes are necessary to inhibit the IP3R

  20. To summarise… Ca2+ Other regulators Ca2+ kinases phosphatases Closed « square » Inactive Open « windmill » IICR 300 [Ca2+] (nM)

  21. Overview 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP3R) 2. Aims 3. 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 (CICR) mechanism in A7r5 and 16HBE014-cells 4. Conclusions and future perspectives

  22. EF1 EF2 EF3 EF4 CaM EF1 EF2 EF3 EF4 Recoverin Family of neuronal Ca2+-binding proteins EF-hand containing Ca2+ 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+ CaBP1 EF1 EF2 EF3 EF4

  23. GST GST 1-604 GST 1-225 (CaM-binding site) GST 226-604 sCaBP1 CaBP1 binds to the IP3R CaBP1 caldendrin lCaBP1 EF1 EF2 EF3 EF4 sCaBP1 Novel agonist of the IP3R?? (Yang et al., 2002)

  24. CaBP1 binds to a similar region of the IP3R as CaM 159 CaM CaM 1 A C D E B F + Ca2+ EGTA sCaBP1 C D F A B E C D F A B E sCaBP1 Binding of CaBP1 to the IP3R is Ca2+-independent

  25. 1000 800 600 Ca2+i (nM) 400 sCaBP1 200 0 lCaBP1 0 200 400 600 800 1000 Time (s) Both Long and Short CaBP1 inhibit IICR 100µM 1µM 0.5µM ATP Intact COS cells Control Decreased amount of responsive cells Increased latency

  26. 45Ca2+ flux on permeabilized cells IP3 binding 100 90 75 70 control 45Ca2+ -release (%) 50 sCaBP1 [3H]IP3 Binding (% vs control) 50 30 25 10 0 10 0,1 1 100 sCaBP1 Ca2+ Ca2+ sCaBP1 control [IP3] (µM) CaBP1 inhibits IICR and IP3 binding CaBP1 acts directly on the IP3R by inhibiting IP3 binding

  27. EF1 EF2 EF3 EF4 Control CaBP1134 CaBP1 inhibits IICR independent of Ca2+ binding YFP 0.5 1 100 µM ATP 1000 CaBP1 activity is regulated by: 800 Myristoylation 600 Ca2+i (nM) Phosphorylation 400 200 0 0 1500 500 1000 Time (s)

  28. CaBP1 does not affect Ca2+ release through RyRs 10µM CCh 1mM Caf 0.5mM Caf 2mM Caf IP3R RyR CaM CaBP1 sCaBP1 To summarise… RyRs are not modulated by CaBPs: In neurons: 300 ) M 200 n ( i ] + 2 a C [ 100 Control 0 0 500 1000 1500 2000 Time (s)

  29. OVERVIEW 1. Introduction Calcium signalling Inositol trisphosphate receptor (IP3R) 2. Aims 3. 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 (CICR) mechanism in A7r5 and 16HBE014-cells 4. Conclusions and future perspectives

  30. New type of Ca2+ -induced Ca2+ release (CICR) IP3 Ca2+ 100 Fractional loss (%/2 min) 50 0 10 20 0 Time (min) 45Ca2+ -flux on permeabilized A7r5 and 16HBE14o-cells Permeabilized 45Ca2+ Loaded Intact ER ER ER CICR is not mediated by IP3R nor RyR EC50: 700 nM

  31. control CaM CaM1234 Effects of CaM and CaM1234 on CICR 40 30 CICR is inhibited by: CaM1234 CaM1 CaBP1 NCS1 Fractional loss (%/ 2 min) 20 10 Ca2+ CaM Ca2+ sensor? 0 10 20 Time (min)

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

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

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

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

  36. To summarise… New type of Ca2+ -induced Ca2+ release channel ?? CICR channel CaM is the Ca2+ sensor Inhibited by CaM mutants Inhibited by CaM-like proteins CaM1234 Mg2+ - - Ca2+ • New type of intracellular Ca2+ channel (Wissing et al, 2002)? • Related to polycystin-2 (Koulen et al., 2002)? • Related to TRPV1 (Liu et al., 2003)? ATP + + CaM IP3R CICR

  37. Conclusions CaM CaBP1 RyR CICR IP3R ER

  38. Future Perspectives • Role of neuronal Ca2+-binding proteins in neurons • Identification of CICR channel

  39. Thank You

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