1. Intracellular calcium release channels as multi-protein complexes Jan B. Parys K.U.Leuven 7 th ECS meeting, June 13th, 2002

2. Intracellular Ca 2+ release channels: IP 3 Rs and RyRs (Furuichi & Mikoshiba, 1995)

3. IP 3 R1 IP 3 R2 IP 3 R3 RyR1 RyR2 RyR3 Intraluminal proteins Calreticulin Calsequestrin Chromogranins A and B Annexin VI ER membrane proteins Triadin Junctin Kinase anchor proteins Plasma membrane proteins Trp’s DHPR G-proteins Cytosolic proteins CaM CaBPs FKBP’s Sorcin S100 IRAG Cytoskeletal proteins Homer Ankyrin Talin Vinculin Alpha-actin Myosin II

4. 1)What is the relation between the IP 3 R and the FKBP-type immunophilins ? 2)What is the relation between the IP 3 R and calmodulin ? 3)What is the relation between the IP 3 R and the cytoskeletal elements ?

5. FKBP12 and FKBP12.6 12-kDa FK506-binding protein Peptidylprolyl isomerase activity (  FK506) Immunophilins (+FK506) inhibit calcineurin Modulators of the IP 3 receptors ? Stabilization of the channel Promotion of coupled gating Modulators of the ryanodine receptors (  FK506) Huse et al., 1999

6. FKBP12- binding site IP 3 R1VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP 3 R2ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP 3 R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV RyR1QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL Y2H

7. FKBP12- binding site IP 3 Ca 2+ FKBP12 NH 2 COOH SI SII POREPORE SIII P P ATP IP 3 R1VCTEGKNVYTEIKCNSLLPLDDIVRVVTHEDCIPEV IP 3 R2ACTEGKNVYTEIKCNSLLPLDDIVRVVTHDDCIPEV IP 3 R3 ACAEGKNVYTEIKCTSLVPLEDVVSVVTHEDCITEV RyR1QAGKGEALRIRAILRSLVPlDDLVGIISLPLQIPTG RyR2HAGKGEAIRIRSILRSLIPLGDLVGVISIAFQMPTI RyR3QTGKGEAIRIRSILRSLVPTEDLVGIISIPLKLPSL Cytosol Lumen of the store

8. FK506 and FKBP12 affect Ca 2+ release through the RyRs but not through the IP 3 Rs BC 3 H1 SH-SY5Y

9. M i c r o s o m e s S o l u b i l i z a t e G S T G S T - F K B P 1 2 Retention of the intracellular Ca 2+ -release channels by GST- FKBP12 affinity assay RyR3 (HEK293) RyR1 (Skel. muscle) Cerebellum: IP 3 R1 RyR1 A7r5: IP 3 R1 IP 3 R3 Sf9: IP 3 R1 IP 3 R3

10. RyR3 IRIRSILRSLVPTEDLVGIISIP RyR3/V2322L IRIRSILRSLLPTEDLVGIISIP (as in IP3R1) RyR3/V2322I IRIRSILRSLIPTEDLVGIISIP (as in RyR2) RyR3/V2322D IRIRSILRSLDPTEDLVGIISIP (negat. charge) RyR3/IP3R1 IRIRSICNSLLPLDDIVGIISIP (as in IP3R1) Mutational analysis of FKBP12-binding site

11. FKBP12 : binding protein binding  stable interaction RyRs : XP residue as an  -helix breaker FKBP12 : enzyme catalysis  transient interaction IP 3 Rs : XP residue in a different secondary structure Putative model

12. RyRs and IP 3 Rs have different FKBP-binding properties FKBP12-binding site on the IP 3 R is much less efficient than the homologous site in the RyR. Higher-order structure may be important. RyRs: stable interaction  FKBP12 is regulatory subunit. IP 3 Rs: weak or transient interaction  chaperone function?  effects on kinetics? FK506  can act on SERCA and on calcineurin.

13. 1)What is the relation between the IP 3 R and the FKBP-type immunophilins ? 2)What is the relation between the IP 3 R and calmodulin ? 3)What is the relation between the IP 3 R and the cytoskeletal elements ?

14. Effects on RyR (Rodney et al., 2001) 3614 3643 ApoCaM: Ca 2+ /CaM: ACTIVATOR OF RYR1INHIBITOR OF RYR1 Ca 2+

15. IP 3 R A7r5 (Missiaen et al., 1999) Effects of CaM on Ca 2+ release:

16. IP 3 R A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca 2+ +10 μM CaM +20 μM CaM Effects of CaM on Ca 2+ release:

17. IP 3 R A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca 2+ +10 μM CaM +20 μM CaM Sf9 (Cardy & Taylor, 1998) Effects of CaM on Ca 2+ release: Effects of CaM on IP 3 binding:

18. IP 3 R A7r5 (Missiaen et al., 1999)Cerebellum (Michikawa et al., 1999) 200 μM Ca 2+ +10 μM CaM +20 μM CaM Sf9 (Cardy & Taylor, 1998) Effects of CaM on Ca 2+ release: Effects of CaM on IP 3 binding: CaM (μM) Lbs-domains (Vanlingen et al., 2000) Lbs-1 Lbs-2 Lbs-3

19. IP 3 Ca 2+ CaM NH 2 COOH SI SII CaM POREPORE SIII Cytosol Lumen of the store P P ATP CaM- binding sites High-affinity Ca 2+ -dependent Types 1 and 2 Low-affinity Ca 2+ -dependent Not in neuronal IP 3 R1 (SII + )

20. Ca 2+ CaM1234 CaM1234 Ca 2+ CaM ApoCaM Ca 2+ Control [ 3 H]IP 3 binding (%) 0.1110 0 20 40 60 80 100 [ 3 H]IP 3 binding (%) Lbs-1 Lbs-1  1-225 1 581226 581 +HIS Effects of CaM on IP 3 binding CaM1234 (μM)

21. Ca 2+ CaM1234 CaM1234 Ca 2+ CaM ApoCaM Ca 2+ Control [ 3 H]IP 3 binding (%) 0.1110 0 20 40 60 80 100 [ 3 H]IP 3 binding (%) Lbs-1 Lbs-1  1-225 1 581226 581 +HIS Effects of CaM on IP 3 binding CaM1234 (μM) IC 50 ~ 2 μM

22. Cyt1 Cyt2 Lbs-1 Lbs-1  1-225 1 581226 581 1159 154309 +HIS +GST Ca 2+ EGTA Detailed localisation of the N-terminal CaM-binding site CaM1234

23. Cyt1 Cyt2 Lbs-1 Lbs-1  1-225 1 581226 581 1159 154 +HIS +GST A B C E D F 1-5-10 1-5-8-14 70% IQ 76% IQ 53% IQ Detailed localisation of the N-terminal CaM-binding site ABCDEF -0.2 0.0 0.2 0.4 0.6 0.8 1.0 200 µM free Ca 2+ 1 mM EGTA 309 Band-shift experiments on non-denaturing gels Interaction with dansyl-CaM

24. Cyt1 Cyt2 Lbs-1 Lbs-1  1-225 1 581226 581 1159 154 +HIS +GST A B C E D F 1-5-10 1-5-8-14 70% IQ 76% IQ 53% IQ Detailed localisation of the N-terminal CaM-binding site ABCDEF -0.2 0.0 0.2 0.4 0.6 0.8 1.0 200 µM Ca 2+ 1mM EGTA 309 Intensity loss (1-B/B o ) K d 0.1 μM K d 1 μM

25. CaM1234 does not inhibit IP 3 -induced Ca 2+ release IP 3 1 μM IP 3 1 μM + CaM 10 μM IP 3 1 μM + CaM1234 10 μM

26. Discontinuous (aa 49-81 and 106-128). Low affinity. Ca 2+ independent. Involved in inhibition of IP 3 binding. Not involved in inhibition of IP 3 -induced Ca 2+ release. Other possible functions: Role in conformation of the IP 3 R ? Tethering of CaM ? Binding site for CaM-like proteins ?  CaBP-1? Protective effect ?  Proteolysis? Oxidative stress? N-terminal CaM-binding site of the IP 3 R

27. 1)What is the relation between the IP 3 R and the FKBP-type immunophilins ? 2)What is the relation between the IP 3 R and calmodulin ? 3)What is the relation between the IP 3 R and the cytoskeletal elements ?

28. Localization of IP 3 R1 and IP 3 R3 in A7r5 smooth-muscle cells IP 3 R1IP 3 R3

29. Redistribution of IP 3 R1 after prolonged stimulation Resting cells+ AVP AVP > 1h Imipramine IP 3 -ester Thapsigargin CPA [Ca 2+ ] cyt

30. 0 20 40 60 80 100 Role of PKC perinuclear IP 3 R1 cytoplasmic IP 3 R1 Percentages of cells with: Cells (%) Control AVP AVP+Staur. TG+Staur. AVP+BIM OAG

31. Role of cytoskeleton 0 10 20 30 40 50 60 70 80 Cells (%) perinuclear IP 3 R1 cytoplasmic IP 3 R1 Percentages of cells with: Control AVP+Nocod. AVP Control AVP+Nocod. AVP Resting cells +AVP Microtubular network

32. Determinants of the localization of IP 3 Rs Various IP 3 R isoforms can have a different distribution. The localization of the IP 3 Rs is dynamic and change with the physiological status of the cell. PKC activation seems an important determinant of the intracellular localization pattern. The microtubular network is involved but which protein(s) determine the IP 3 R localization is not yet ascertained.

33. Conclusions  The intracellular Ca 2+ release channels IP 3 Rs and RyRs act as part of a multiprotein complex.  Many proteins have been proposed as partners.  The existence of consensus binding sites is not sufficient for assuring binding in vivo. Higher-order structure, experimental conditions and indirect associations can be involved.  RyRs and IP 3 Rs are regulated by different proteins.  Future work on the IP 3 R will focus on the interactions with the various Ca 2+ -binding proteins and with the different scaffolding proteins.

34. Geert Callewaert Humbert De Smedt Ludwig Missiaen Jan B. Parys IP 3 -team (Leuven, Belgium) Geert Bultynck Patrick De Smet Nael Nadif Kasri Ilse Sienaert Karolina Szlufcik Kristel Van Acker Sara Vanlingen Elke Vermassen

35. Geert Callewaert Humbert De Smedt Ludwig Missiaen Jan B. Parys IP 3 -team (Leuven, Belgium) Geert Bultynck Patrick De Smet Nael Nadif Kasri Ilse Sienaert Karolina Szlufcik Kristel Van Acker Sara Vanlingen Elke Vermassen