Regulation of the IP3 Receptor by Ca2+ and Ca2+-Binding Proteins Laboratory of Physiology KULeuven Leuven, Belgium

R LGCC VGCC SOCC MSCC NCX IP3R RYR NCX PMCA SERCA Others?? ER/SR PMR1 Second messenger IP3R RYR NCX Mitochondria PMCA Others?? IP3R ER/SR PMR1 SERCA Uniporter golgi Buffers

Agonists IP3 Ca2+ IP3R I, II, III Cytoskeletal proteins: Actin; MyosinII Ankyrin; Tallin; Vinculin 4.1N Agonists IP3 Ca2+ Plasma membrane associated: Homer-mGluR TRPs; RhoA-TRPC1 G Kinases and phosphatases: PKA; Fyn BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1 Cytosolic proteins: Calmodulin; CaBP IRBIT; CARP IP3R I, II, III Intraluminal proteins: Chromogranins; Calnexin, Calreticulin

Regulation of the IP3R by Ca2+ Structure of the IP3R Bell-shaped IICR Ca2+ is the primary modulator of its own release by intracellular Ca2+ release channels

Effect of Calmodulin on IP3-induced Ca2+ release A7r5 cells 70% IP3R1 CaM Control HBE cells 90% IP3R3 Control CaM A7r5 HBE

Calmodulin and apocalmodulin Sienaert, I,. Nadif Kasri, N, Vanlingen S., Parys J. B., Callewaert G., Missiaen, L., and De Smedt, H. Localisation and Characterisation of a calmodulin/ apocalmodulin binding domain in the N-terminal part of th etype 1 inositol1,4,5- trisphosphate receptor Biochem. J. 365, 269-277, 2002

Lbs-1: IP3 binding core (226-604) Lbs-1  1-225 (Adkins et al., 2000) CaM NH2 CYT Ca2+CaM COOH ER Recombinant ligand-binding domain of IP3R1 (LBS-1) 1 581 kDa 62 – 49 38 28 17 14 6 Lbs-1 1-225 W226 581 Lbs-1  1-225

Calmodulin effect on IP3 binding [3H]IP3 binding (%) Lbs-1His Lbs-1  1-225His 1 581 W226 Ca2+ Control CaM1234 Ca2+/CaM1234 Ca2+/CaM CaM EC50= 1.7 µM 5 10 0.0 0.1 0.2 0.3 B/F Bound (nM) CaM inhibits IP3 binding in Ca2+ -independent way

GST-fusion protein pull down Localisation of a Calmodulin-Binding Site GST-fusion protein pull down of CaM1234 CaM1234 pGST GST-Cyt1 GST-Cyt2 50 M free Ca2+ 1 mM EGTA Cyt1 Cyt2 Lbs-1 Lbs-1  1-225 1 581 W226 159 309

Detailed localisation using peptides A B C D E F CaM A B C E D F 1 159 1-5-10 1-5-8-14 70% IQ (site1) 76% IQ 53% IQ A B C D E F CaM Ca2+ EGTA 1- B/Bo Ca2+ independent CaM-binding site in the N-terminal Region

Calmodulin binding sites on IP3R1 R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD 13 18 31 25 Endoplasmic reticulum Cytosol CaM W1577A (Zhang et al, 2001; Nosyreva et al, 2002) R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA Ca2+/CaM

IICR is inhibited by CaM and CaM1234 Control 0.5 1 100 600 Control CaM 400 Ca2+i (nM) 200 250 500 750 1000

Regulation of Calcium release neuronal Calcium Binding Proteins (CaBP) By neuronal Calcium Binding Proteins (CaBP) Regulation of Calcium release by neuronal Calcium Binding Proteins (CaBP) N. Nadif Kasri, H. Llewelyn Roderick, J.B. Parys, L. Missiaen, H. De Smedt and M. Bootman In preparation

CaM or other CaM-like Ca2+ sensor proteins ? Adapted from Haeseleer et al., 2000 Inhibitory Activatory ? (Yang et al., 2002)

CaBP binds to the InsP3R CaBP GST 1-604 1-225 226-604 NH2 COOH ER CYT IP3 binding core (226-604) CaBP GST 1-604 1-225 226-604

CaBP binds to a similar region of the InsP3R as CaM 1 159 CaM sCaBP1 + Ca2+ -Ca2+/ EGTA A) B) CaBP binds to a similar region of the InsP3R as CaM

Ratio of CaBP: peptide B Binding of CaBP to the InsP3R is calcium independent Ratio of CaBP: peptide B CaBP CaBP 1/10 1/1 1/2 1/3 1/4 1/5 1/6 1/8 + Calcium + EGTA 2 4 6 8 10 0.0 0.5 1.0 Band intensity Peptide B: CaBP

CaBP inhibits InsP3 binding to the InsP3R 4 µg Lbs-1 10 µM sCaBP1 5 µM Ca2+ Lbs-1 sCaBP1 Ca Ca/ 100 [3H]IP3 Binding (% vs control) 100% 78 ± 1.9 % 68 ± 6.9 % 39 ± 4.7% 75 50 25 CaBP inhibits InsP3 binding to the InsP3R

Both Long and Short CaBPs inhibit InsP3 induced Calcium release Control 200 400 600 800 1000 SCaBP LCaBP 0.5µM 1µM 100µM ATP Time (s) Ca2+i (nM) Both Long and Short CaBPs inhibit InsP3 induced Calcium release

CaBP inhibits InsP3 induced Calcium release independent of Calcium binding EF1 EF2 EF3 EF4 1000 800 600 400 200 1500 500 Time (s) Ca2+i (nM) 0.5 1 100 µM ATP Control 100 50 % responsive cells 0.5 1 ATP (mM) CaBP134

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

Summary CaBP binds on the N-terminal CaM-binding site in a Ca2+-independent way CaBP inhibits IP3 binding CaBP inhibits IICR CaBP activity is Calcium independent

?? 225-604 GST Control IP3 AdPhos CaM sCaBP-1 1-225

Suramin Interacts with the CaM-binding sites on the IP3R1 Control 100 µM suramin IP3R1 Input Seph CaM-Seph + Suramin EGTA Ca 2+ Control 10 µM CaM 1-225 EGTA Ca 2+

MLCK peptide inhibits IICR High CaM affinity binding properties IP3

Properties of the inhibition of IICR by MLCK peptide Inhibition of IICR by other CaM-binding peptides? RyR pep: 10 nM IP3 Trp3C14: 100 nM IP3

Summary Suramin interacts with the CaM-binding sites on the IP3R Suramin Inhibits IICR by lowering the IC50 for activation by IP3 MLCK peptide inhibits IICR by lowering Vmax By removing endogenous CaM? By disrupting intermolecular protein-protein interaction?

Conclusions Ca2+ is the major regulator of the IP3R CaM inhibts IICR only in the presence of Ca2+ Two CaM-binding sites on the IP3R Ca2+ dependent in the regulatory domain Ca2+ independent in the N-terminus CaM can play an important role in intramolecular protein-protein interactions within IP3R, as removing endogenous CaM inhibits IICR

A new Calmodulin-dependent CICR mode in A7r5 cells N. Nadif Kasri, I. Sienaert, Jan B. Parys, G. Callewaert, L. Missiaen, A. Jeromin and H. De Smedt A novel Ca2+-induced Ca2+-release mechanism in A7r5 cells regulated by calmodulin-like proteins J. Biol. Chem, 2003 in the press

45Ca2+-efflux technique on permeabilized A7r5 cells Culture medium Saponin 45Ca2+ TG SDS Fractional loss

Ca2+ -induced Ca2+ Release in A7r5 cells Ca2+ release vs control CICR is not mediated via IP3R or RyR Control Heparin 2-APB XeC RuRed Ryanodine

Characteristics of the CICR mode Ca2+ dependence: EC50 = 700 nM Hill = 1.9 Mg2+ inhibition: EC50= 0.6 mM ATP stimulation: EC50= 320 µM

Effects of CaM, CaM1 and CaM1234 40 30 20 control 10 10 20 Time (min) control CaM1 CaM CaM1234

Calmodulin Calmodulin Calmodulin Calmodulin1 Calmodulin1 Calmodulin1234 Long CaBP1 NCS - 1/Frequenin Short CaBP1 1/FrequeninE120Q Calmodulin1234 Calmodulin1234 Long CaBP1 Long CaBP1 Short CaBP1 Short CaBP1 NCS - 1/Frequenin NCS NCS - - 1/Frequenin 1/Frequenin NCS NCS - - 1/FrequeninE120Q 1/FrequeninE120Q

Preincubation with a CaM-binding peptide inhibits CICR control RyR CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

CaM but not CaM1234 can restore CICR Preincubation with RyR CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

CaM but not CaM1234 can restore CICR Preincubation with RyR CaM-BS (peptide aa 3614-3643) CaM CaM1234 Ca2+ (3 µM)

Summary Novel CICR mechanism in A7r5 cells IC50: 700 nM Activated by ATP Inhibited by MgCl2 CICR mechanism is regulated by CaM

224 576 PIP2 IP3 Gα PLC 2749 2275

Lab. of Physiology K.U.L., Belgium Brabaham Institute, Cambridge, UK Ilse Sienaert Humbert De Smedt Geert Callewaert Ludwig Missiaen Jan B. Parys Karolina Szlufcik Geert Bultynck Sarah Vanlingen Llewelyn Roderick Martin Bootman Baylor College of Medicine, Houston, Texas Andreas Jeromin