IP3-induced Ca2+ release and calmodulin Laboratory of Physiology KULeuven Leuven, Belgium

Agonists IP3 Ca2+ IP3R I, II, III Plasma membrane associated: 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 HAP1A-Htt IP3R I, II, III Intraluminal proteins: Chromogranins; Calnexin

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

CaM at N-terminal site inhibits IP3 binding Lbs-1: IP3- binding core (226-581) aa 1-225 NH2 COOH ER CYT CaM Adkins et. al., 2000 Ca2+CaM Yamada et. al., 1995 Lin et. al., 2000 Ca2+CaM SII Recombinant ligand-binding domain of IP3R1 (LBS-1) Lbs-1 Lbs-1  1-225 1 581 W226 1-225

The inhibition is Ca2+ independent EC50= 1.7µM Lbs-1His Lbs-1  1-225His 1 581 W226 Ca2+ CaM1234 10 µM CaM1234 Ca2+ CaM 10 µM apoCaM 5 µM Ca2+ control [3H]IP3 binding (%) 5 10 0.0 0.1 0.2 0.3 B/F Bound (nM)

CaM-binding sites in the N-terminal region are Ca2+ independent 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+ A B C D E F CaM EGTA Ca2+ independent EC50  1-1.5 µM

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

In permeabilized L15 cells IICR is inhibited by CaM and by CaM1234 L15 cells % 40 Ca2+ release vs A23187 (%) 50 60 70 80 90 100 IP3R1 IP3R3 control CaM CaM1234 1 2 3 4 5 [Ca2+] µM CaM is not the Ca2+ sensor for the inhibition of IICR

Endoplasmic reticulum Cytosol CaM The Ca2+/CaM site in the coupling region is not involved in the CaM inhibition of IICR 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

CONCLUSION: The N-terminal Ca2+-independent CaM-binding site is responsible for the CaM inhibition of IICR Significance ? 1) Interaction site for other CaM-like proteins ? 2) Involved in intramolecular interactions?

CaM or other CaM-like Ca2+ sensor proteins ? Interaction with neuronal Calcium Binding Proteins (CaBP) Adapted from Haeseleer et al., 2000 Inhibitory Activatory ? (Yang et al., 2002)

CaBP1 binds to the N-terminal part of the IP3R NH2 COOH ER CYT IP3 binding core (226-581) aa 1-225 CaM CaBP1 ? Ca2+CaM CaBP1 GST 1-581 1-225 226-581

CaBP1 binds to a similar region as CaM independently of Ca2+ A) 1 CaM CaM 159 A C D B E F B) + Ca2+ -Ca2+/ EGTA sCaBP1 C D F A B E sCaBP1 C D F A B E

Both long and short CaBPs inhibit IP3-induced calcium release in COS-1 cells 45Ca2+ flux Control 200 400 600 800 1000 sCaBP lCaBP 0.5µM 1µM 100µM ATP Time (s) Ca2+i (nM)

CaBP inhibits IP3-induced Ca2+ release independent of Ca2+ binding EF1 EF2 EF3 EF4 YFP 1000 800 600 400 200 1500 500 Time (s) [Ca2+ ]i (nM) 0.5 1 100 µM ATP Control CaBP134

Significance ? 1) Interaction site for other CaM-like proteins ? 2) Involved in intramolecular interactions?

Suramin interacts with the CaM-binding sites on IP3R1 Suramin mimics the CaM inhibition of IICR Control 100 µM suramin 1-225 EGTA Ca 2+ IP3R1 Input Seph CaM-Seph + Suramin Control 10 µM CaM

N-terminal CaM-binding site = intramolecular interaction site? Suramin CABP1 CaM1234 Interaction 1-225 with IBC GST / IP3 AdPhos CaM sCaBP-1 IP3 binding core (IBC) CaM NH2 COOH ER CYT 1-->225 Ca2+CaM

In permeabilized A7r5 cells Suramin induced a large IP3-independent Ca2+ release

New type of Ca2+ -induced Ca2+ release (CICR) channel ?? + CaM1234

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 Fractional loss (%/ 2 min) 10 10 20 Time (min) control CaM1 CaM CaM1234

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

Preincubation with a CaM-binding peptide inhibits CICR control RyR1 CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) CaM Ca2+ (3 µM)

CaM but not CaM1234 can restore CICR Preincubation with RyR1 CaM-BS (peptide aa 3614-3643) CaM1234 CaM Ca2+ (3 µM)

New type of Ca2+ -induced Ca2+ release channel ?? CONCLUSIONS Mg2+ - CaM1234 Inhibited by CaM mutants Inhibited by CaM-like proteins CICR channel Ca2+ + ATP suramin CaM is the Ca2+ sensor CaM IP3R CICR New type of intracellular Ca2+ channel (Wissing et al, 2002)? Related to polycystin-2 (Koulen et al., 2002)? Related to TRPV1 (Liu et al., 2003)? Truncated IP3R?

Baylor College of Medicine Houston, Texas KULeuven Leuven, Belgium Babraham Institute Cambridge UK Jan B. PARYS Geert CALLEWAERT Ludwig MISSIAEN Rafael A. FISSORE Nael NADIF KASRI Geert BULTYNCK Karolina SZLUFCIK Leen VERBERT Elke VERMASSEN Zerihun ASSEFA Iris CARTON Patrick DE SMET H. Llewelyn Roderick Martin D. Bootman Michael Berridge Baylor College of Medicine Houston, Texas Andreas Jeromin The Division of Molecular Neurobiology The Institute of Medical Science The University of Tokyo Katsuhiko Mikoshiba