1. Regulation of inositol 1,4,5- trisphosphate (IP 3 ) receptors and IP 3 -induced Ca 2+ release H. De Smedt K.U.Leuven, Belgium

2. IP 3 -binding domain CC NN Ca 2+ Transmembrane region ER Luminal loop Regulatory domain cytosol IP 3 R1: 3D reconstruction (Hamada et al., 2003) Bar: 10 nm

3. Agonists IP 3 Ca 2+ Cytoskeletal proteins: Actin; MyosinII Ankyrin; Tallin; Vinculin 4.1N Cytosolic proteins: Calmodulin; CaBP IRBIT, CARP HAP1A-Htt Cyt C, Bcl-2, BclXL Plasma membrane associated: Homer-mGluR TRP; RhoA-TRPC1 G  ; RACK-1 Kinases and phosphatases: PKA; IRAG-PKG Fyn, BANK- PTK FKBP12-Calcineurin PP1  Intraluminal proteins: Chromogranins; Calnexin CC NN Ca 2+ ER xx Caspase-3

4. IICR Part 1: Regulation of the IP 3 R by CaM + CaM (Missiaen et al., 1999) [Ca 2+ ] (nM) 300 -CaM (Michikawa et al., 1999) IP 3 R I II III IP 3 R I II III [IP 3 ] (µM) + CaM

5. Calmodulin-binding sites on IP 3 R1 13 Endoplasmic reticulum Cytosol R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA CaCaM R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD Ca 2+ -indep. CaM W1577A (Zhang et al, 2001; Nosyreva et al, 2002)

6. 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 Both Ca 2+ and CaM bind to the suppressor and inhibit IP 3 binding but CaM is not the Ca 2+ sensor

7. 0.5 µM1 µM100 µM Control CaM IICR is inhibited by CaM, CaM 1234 and CaBP1 Time (s) ATP 600 400 200 Ca 2+ i (nM) 1000750500250 0 0 CaM 1234 Intact COS cells CaBP1

8. CaM-binding sites on the suppressor domain (199) (200) 1-225 T1 1-225 T3 1-225 T1 1-225 T3 1-225 T1 1-225 T3 1-225 T1 1-225 T3 1-225 T1 1-225 T3 Bosanac et al., Mol. Cell, 2005 76 86 75 87

9. Exchange or deletion of the less conserved region in the arm subdomain of the suppressor is critical for IP 3 sensitivity

10. CaM-binding sites on the suppressor domain (199) (200) 1-225 T1 1-225 T3 1-225 T1 1-225 T3 1-225 T1 1-225 T3 1-225 T1 1-225 T3 1-225 T1 1-225 T3 Bosanac et al., Mol. Cell, 2005 CaM-binding CaM- and CaBP1-binding

11. Depletion of CaM with high-affinity CaM-binding peptides uncouples IICR from IP3 binding

12. Depletion of endogenous CaM by MLCK peptide inhibits IICR in permeabilized L15 fibroblasts EGTA 0.5 µM Ca 2+ Ac-RRKWQKTGHAVRAIGRL-NH2 (Kd 6 pM) control 0.5 µM Ca 2+ Ac-RRKEQKTGHAVRAIGRE-NH2

13. MLCK peptide does not change the affinity for IP 3 10 µM CaM Control 1 µM MLCK 10 µM MLCK

14. Inhibition of IICR by different CaM-binding peptidesis dependent on their affinity for CaM

15. The inhibition by MLCK peptide can be reversed by Ca 2+ -CaM but not by CaM 1234 MLCK pep CaM IP 3 CaM 1234 CaM

16. Regulation via CaM and Ca 2+ CaM sites 13 Endoplasmic reticulum Cytosol ATP P P P P CaCaM NH 2 COOH IP 3 Ca 2+ sensor P 1852S Ca 2+ -indep CaM Depletion of constitutive CaM ?

17. Part 2: The IP 3 R as a Ca 2+ -leak pathway;Truncation by caspase-3 13 Endoplasmic reticulum Cytosol ATP P P P P CaCaM NH 2 COOH IP 3 Ca 2+ sensor Ca 2+ -indep CaM KKDDEVDRDA Caspase-3 cleavage P

18. Caspase-3 mediated cleavage of IP 3 R1 in a DT40 triple(IP 3 R)-KO background Caspase-3 activityApoptosis

19. Aged mouse eggs mouse eggs upon injection of channel only domain mRNA The role of a leaky IP 3 R in mouse oocytes Perturbed Ca 2+ oscillations

20. Overexpression of PS1/PS2 mutants → potentiation IP 3 -mediated Ca 2+ release → increased IP 3 sensitivity → suppression of Capacitive Ca 2+ Entry (CCE) PS1/PS2 knockouts → decreased IP 3 -mediated Ca 2+ release → potentation of CCE Altered intracellular Ca 2+ homeostasis in Alzheimer’s disease Role of presenilins (PS) Hypothesis: altered ER Ca 2+ content? (LaFerla et al., Nature, 2003)

21. CaCl 2 WT PS DKO WT PS1-Rescue Presenilin (PS1/PS2) knockout decreases the [Ca 2+ ] ER as measured by ER-targeted aequorin How does Presenilin alter the [Ca 2+ ] ER ?

22. BiP CRT SERCA IP 3 R1 IP 3 R3 IP 3 R1 IP 3 R3 WT PS DKO Rescue Expression of proteins involved in Ca 2+ handling WT PS DKO Rescue IP 3 R1 IP 3 R3 IP 3 R1 IP 3 R3

23. Basal Ca 2+ leak is increased in PS DKO cells Permeabilized cellsIntact cells WT PS DKO Rescue

24. WT PS DKO PS DKO RNAi PS DKO RNAi control IP 3 R1 IP 3 R3 IP 3 -induced Ca 2+ release vs A23187 RNA i knock-down of IP 3 R1 restored IICR in PS DKO cells to control values WT PS DKO PS DKO PS DKO RNAi RNAi control

25. IP 3 R1 knock-down by RNAi restored Ca 2+ content and Ca 2+ leak in PS DKO cells to control values Ca 2+ content (fmol/cell) WT PS DKO PS DKO; IP 3 R1-RNAi PS DKO RNAi control WTPS DKO IP 3 R1-RNAi PS DKO RNAi Control

26. Why is IP 3 R1 ‘leaky’ ? The Ca 2+ leak is not IP 3 dependent PS DKO PS DKO + heparin Hyperphosphorylated IP 3 R (Oakes et al., PNAS 2005 ) or enhanced Bcl-2 expression are not responsible for leak in PS DKO cells Bcl-2 wt PS DKO P-Ser IP 3 R1 95kDa wtPS DKO Full size IP 3 R1 There is significant cleavage of the IP 3 R with formation of a channel-only domain

27. 1)Cleavage of the IP 3 R in a position upstream of the channel domain creates an IP 3 -independent Ca 2+ channel 2)The IP 3 R behaves as an IP 3 -independent Ca 2+ -leak channel in presenilin-knockout cells Part 2: The IP 3 R as an IP 3 -independent Ca 2+ -leak channel

28. Jan B. PARYS - Ludwig MISSIAEN Humbert DE SMEDT IP 3 -team (Leuven, Belgium) Zerihun ASSEFA Geert BULTYNCK Sarah KOCKS Nael NADIF KASRI Karolina SZLUFCIK Veerle VANDERHEYDEN Leen VERBERT Benoit DEVOGELAERE In collaboration with the groups of: A. Galione (University of Oxford) K. Török (St George’s University of London) R.A. FISSORE (Univ. Massachusetts) M.J. BERRIDGE – M.D. BOOTMAN – L. RODERICK (Babraham) B. DE STROOPER (Genetics – K.U.Leuven) G. CALLEWAERT (Physiology – KULAK)