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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 K.U.Leuven. Intracellular calcium homeostasis. Ca 2+ channels. NCX. IP 3 R. RyR. PMCA. IP 3 R. SPCA1. SERCA. Uniporter. Ca 2+ buffers/sensors. [Ca 2+ ]~ 1 mM.

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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 K.U.Leuven

  2. 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

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

  4. 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) IRBIT IP3 Agonists IP3and Ca2+ COOH 13 ER ERp44 CRT/CNX (Higo et al. 2005, Cell)

  5. β β IP3 α IP3R is divided into 5 parts: Gatekeeper domain Modulatory domain N C Transmembrane domain SI SIII SII N-terminal ligand binding domain Suppressor β α IP3 binding core 578 1 225 226 Closure of the cleft α

  6. 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

  7. Regulation of the IP3R by Ca2+ and calmodulin

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

  9. 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

  10. 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 ER

  11. 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

  12. Localisation of the N-terminal calmodulin-binding site GST-fusion protein pull down of CaM1234 pGST GST-Cyt1GST-Cyt2 50 M free Ca2+ CaM1234 1 mM EGTA CaM1234 1 581 IP3 binding core suppressor 226 581 IP3 binding core 309 159 1 Cyt1Cyt2

  13. 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

  14. Both sites essential? 1 581 IP3 binding core 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. To summarise… Ca2+ Other regulators Ca2+ kinases phosphatases Closed « square » Inactive Open « windmill » IICR Vermassen et al., 2004,BBRC 300 [Ca2+] (nM)

  20. Regulation of intracellular Ca2+ release by neuronal Ca2+-binding proteins

  21. 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

  22. Why so many CaBPs….? Cellular localisation Ca2+ myristyol switch * * * Golgi High Ca2+ Low Ca2+ * * NUCLEUS Burgoyne et al., 2004

  23. Exocytosis in Synapses Exocytosis in Endocrine cells Basal Ca2+ Channel regulation 100 80 NCS1 VILIP-1 CaM Synaptotagmin 60 Ca2+ bound 40 20 0 7 6 5 4 3 -log[Ca2]+ Why so many CaBPs….? Cellular Localisation and…. Adapted from Burgoyne and Weiss 2001

  24. 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)

  25. 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

  26. EF1 EF2 EF3 EF4 CaBP1 SCaBP1 LCaBP1

  27. 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

  28. 30 20 10 0 5 1.5 3 4 1.0 2 Integrated Calcium response nMsx10 -4 3 2 0.5 1 1 0 0 0 0.5 µM ATP 1 µM ATP 100 µM ATP SCaBP1 (n=22) LCaBP1 (n=17) Control (n=44) 100 * 80 * 60 * Latency (s) Responsive Cells 40 * 20 0 0.5 µM ATP 1 µM ATP

  29. SCaBP1 CaBP overexpression inhibits InsP3 Ester induced Calcium release: CaBP acts directly on the InsP3R 10 mM InsP3 ester 250 200 Control 150 Ca2+i (nM) 100 50 0 0 200 400 600 800 1000 1200 time (s)

  30. 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

  31. EF1 EF2 EF3 EF4 Control CaBP1134 CaBP1 inhibits IICR independent of Ca2+ binding YFP 0.5 µM 1 µM 100 µM ATP 1000 800 600 Ca2+i (nM) 400 200 0 0 1500 500 1000 Time (s)

  32. To summarise.. • IICR is inhibited by CaBP • CaBP binds to the receptor in a calcium independent manner resulting in inhibition of IP3 binding to its receptor. CaBP does not activate calcium release as previously reported But, how is CaBP activity regulated?

  33. Membrane Targeting of CaBP may regulate its activity (1) SCaBP1-G2A LCaBP1-G2A SCaBP1 LCaBP1

  34. Membrane Targeting of CaBP may regulate its activity (2) * * 100 % responsive cells 50 0 ATP (mM) 0.5 1 100 sCaBP -G2A 900 * 450 Peak amp (nM) 0 ATP (mM) 0.5 1 100 1 100 0.5 ATP (µM ) 1000 750 Control Ca2+i (nM) 500 250 0 1500 1000 0 500 time (s)

  35. IP WB wt wt YFP S120A YFP S120A 32P- CaBP 0.5 1 100 YFP-CaBP ATP (µM) YFP * * 100 1200 Control % responsive cells 50 CaBP DCK2 Ca2+i (nM) 800 0 0.5 1 100 * ATP (µM) 400 * * 1500 0 1000 500 0 time (s) 900 Peak amplitude (nM) 450 0 0.5 1 100 ATP (µM) Phosphorylation of Serine 120 regulates CaBP activity

  36. StsE (S120~S101 in CaM)

  37. CaBP1 does not affect Ca2+ release through RyRs 10µM CCh 1mM Caf 0.5mM Caf 2mM Caf 200 * Ca2+i (nM) 100 0 2mM Caf 1mM Caf 0.5mM Caf 10µM CCh sCaBP1 300 ) M 200 n ( i ] + 2 a C [ 100 Control 0 0 500 1000 1500 2000 Time (s)

  38. Summary • CaBP inhibits IICR • CaBP binds to the InsP3R inhibiting IP3 binding • CaBP activity is Calcium independent • Myristoylation is not necessary for CaBP activity, although it may have a regulatory role • Phosphorylation of CaBP increases it’s potency • RyRs are not modulated by CaBP • CaBPs may bind in preference to CaM to the InsP3R in neuronal cells regulating Calcium release

  39. A novel Ca2+ -induced Ca2+ release (CICR) mechanism in A7r5 and 16HBE014-cells

  40. 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 Ca2+

  41. 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)

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

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

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

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

  46. To summarise… New type of Ca2+ -induced Ca2+ release channel ?? CICR channel CaM1234 CaM is the Ca2+ sensor Mg2+ - Inhibited by CaM mutants Inhibited by CaM-like proteins - Ca2+ ATP + + CaM IP3R CICR

  47. Conclusions CaBP1 CaM Low [Ca2+] High [Ca2+] - + + - - RyR CICR IP3R ER

  48. Geert BULTYNCKSarah KOCKSElke VERMASSEN Karolina SZLUFCIKVeerle VANDERHEYDENLeen VERBERTBenoit DEVOGELAERE IP3-team (Leuven, Belgium) Geert CALLEWAERT - Jan B. PARYS - Ludwig MISSIAEN Humbert DE SMEDT In collaboration with the groups of:K. MIKOSHIBA (Univ. Tokyo)R.A. FISSORE (Univ. Massachusetts)M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham)

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