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

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

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

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

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)

C N IP 3 binding core 581 IP 3 binding core suppressor 1 Control [ 3 H]IP 3 binding (%) 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

0.5 µM1 µM100 µM Control CaM IICR is inhibited by CaM, CaM 1234 and CaBP1 Time (s) ATP Ca 2+ i (nM) CaM 1234 Intact COS cells CaBP1

CaM-binding sites on the suppressor domain (199) (200) T T T T T T T T T T3 Bosanac et al., Mol. Cell,

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

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

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

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

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

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

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

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 ?

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

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

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

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)

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 ?

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

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

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

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

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

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

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)