Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -binding Proteins
Introduction IP 3 R Regulation of the IP 3 R by Ca 2+ and CaM Results Localization and characterization of the CaM-binding sites Function of CaM Regulation of the IP 3 R by Neuronal Ca 2+ -binding proteins Conclusions
IP 3 R I, II, III Agonists IP 3 Ca 2+ Intraluminal proteins: Chromogranins; Calnexin, Calreticulin Cytosolic proteins: Calmodulin; CaBP IRBIT; CARP Cytoskeletal proteins: Actin; MyosinII Ankyrin; Talin; Vinculin 4.1N Plasma membrane associated: Homer-mGluR TRPs; G Kinases and phosphatases: PKA; PKC; Fyn BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1 ER cytosol
Ca 2+ is the primary modulator of its own release Structure of the IP 3 R Regulation of the IP 3 R by Ca 2+ Bell-shaped IICR Hamada et al., 2002 Bezprozvanny et al., 1991
IICR Regulation of the IP 3 R by CaM + CaM (Missiaen et al., 1999) [Ca 2+ ] (nM)300 - CaM (Michikawa et al., 1999) CaM Ca 2+ dependently inhibits IP 3 R I, II and III IP 3 R I II III
Calmodulin binding sites on IP 3 R1 13 Endoplasmic reticulum Cytosol R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA CaCaM CaM (Adkins et al,2000) W1577A (Zhang et al, 2001; Nosyreva et al, 2002)
Localization and characterization of the CaM-binding sites Results
Calmodulin effects on IP 3 binding CaM inhibits IP 3 binding in Ca 2+ -independent way C N B/F Bound (nM) [ 3 H]IP 3 binding (%) Control CaM Ca 2+ /CaM CaM 1234 Ca 2+ /CaM 1234 Ca 2+ IP 3 binding core
Localisation of a calmodulin-binding site GST-fusion protein pull down of CaM 1234 CaM 1234 pGST GST-Cyt1 GST-Cyt2 50 M free Ca 2+ 1 mM EGTA CaM 1234 Cyt1 Cyt IP 3 binding core
Detailed localisation using peptides A B C D E F CaM A B C E D F % IQ 76% IQ 53% IQ A B C D E F CaM Ca 2+ EGTA Discontinue Ca 2+ -independent CaM-binding site in the N- terminal region
A B C E D F 1159 control∆ B∆ E [ 3 H]IP 3 binding Both CaM-binding sites are essential for inhibiting IP 3 binding CaM
Calmodulin-binding sites on IP 3 R1 13 ER Cytosol R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD CaCaM CaMCa 2+
0.5 µM1 µM100 µM Control CaM IICR is inhibited by CaM and CaM 1234 Time (s) ATP Ca 2+ i (nM) CaM 1234 CaM CaM 1234 Control
CaM is not the Ca 2+ sensor for the IP 3 R 200 nM IP 3 CaM 1234 CaM [Ca 2+ ] (µM) 45 Ca 2+ flux 0.11 IICR is inhibited by CaM and CaM Ca 2+ release (%)
Half CaM’s CaM C-CaM N-CaM 3 HIP 3 binding control 45 Ca 2+ flux control Both CaM lobes are necessary to inhibit the IP 3 R
Ca 2+ closed open IICR [Ca 2+ ] (nM) 300 inactive Other regulators PKC? The N-terminal Ca 2+ -independent CaM-binding Site on the IP 3 R is Responsible for CaM Inhibition, even though this Inhibition Requires Ca 2+
Summary Two CaM-binding sites on the IP 3 R Ca 2+ dependent in the regulatory domain Ca 2+ independent in the N-terminus CaM inhibits IICR only in the presence of Ca 2+ CaM inhibits IICR by lowering the affinity for IP 3, CaM is not the Ca 2+ sensor
1-225 EGTACa 2+ IP 3 R1 input - Seph CaM-Seph Seph Suramin +-+- Suramin interacts with the CaM-binding sites on the IP 3 R1 (1) Suramin: polysulfonated naphthylurea Interacted with the RYR CaM-binding sites (papineni et al., 2002)
Suramin interacts in a direct way with the CaM-binding sites Suramin CaM BE B E 0 µM100 µM0 µM100 µM Suramin interacts with the CaM-binding sites on the IP 3 R1 (2)
Suramin inhibits IICR Does Suramin counteract the effects of CaM? IP 3 R1IP 3 R3 suramin Contr ol 10 µM CaM Missiaen et al., 1999 Suramin inhibits IICR
IP 3 binding core Suramin [ 3 H]IP 3 Binding Suramin inhibits IP 3 binding [ 3 H]IP 3 Binding [Suramin] (µM) B/F Bound (nM) 05 suramin
Summary Suramin inhibits IICR by lowering the IC 50 for activation by IP 3 Suramin interacts with the CaM-binding sites on the IP 3 R Suramin is a potent inhibitor of IICR Suramin mimicks the effects of CaM
Regulation of calcium release by neuronal Calcium Binding Proteins (CaBP)
CaM CaM-like L-CaBP1 S-CaBP1 Caldendrin S-CaBP5 S-CaBP2 L-CaBP2 S-CaBP3 CaBP3 GCAP GCAP3 GCAP2 Recoverin Visinin Neurocalcin VILIP3 VILIP1 VILIP2 NCS1 EF1EF2EF3EF4 CaM EF1EF2EF3EF4 CaBP1 EF1EF2EF3EF4 CaBP2 EF3EF4 CaBP3 EF1EF2EF3EF4 CaBP4 EF1EF2EF3EF4 CaBP5 Adapted from Haeseleer et al., 2000 CaBP NCS
NUCLEUS High Ca 2+ Golgi Why so many CaBPs….? Cellular localisation Ca 2+ myristyol switch NUCLEUS Golgi Low Ca 2+ Burgoyne et al., 2004
Exocytosis in Endocrine cells Exocytosis in Synapses Channel regulation Basal Ca2+ NCS1VILIP-1CaMSynaptotagmin Ca 2+ bound -log[Ca 2 ] + Adapted from Burgoyne and Weiss 2001 Why so many CaBPs….? Cellular Localisation and….
CaBPs agonist of the IP 3 R? Taken from Yang et al., 2002 CaBPs are agonists of the IP 3 R CaBPs bind to N-terminus of the IP 3 R CaBPs binding is Ca 2+ dependent CaBPs co-localize with the IP 3 R
sCaBP1 GST GST GST CaBP1 binds to the IP 3 R NH 2 COOH ER CYT IP 3 binding core ( ) caldendrin lCaBP1 sCaBP1
A B C E D F CaM sCaBP1 ABCDEF ABCDEF + Ca 2+ EGTA CaBP1 binds to a similar region of the IP 3 R as CaM
CaBP 1/11/21/41/5 1/6 1/8 1/10 1/3 CaBP Ratio of CaBP: peptide B Band intensity Peptide B: CaBP + Calcium + EGTA Binding of CaBP to the IP 3 R is Ca 2+ independent
EF1EF2EF3EF4 CaBP1 lCaBP1 sCaBP1 Cellular localization of CaBP1
sCaBP 0.5µM 1µM 100µM ATP Control lCaBP Time (s) Ca 2+ i (nM) Both Long and Short CaBP1 inhibit IICR
SCaBP1 (n=22) LCaBP1 (n=17) Control (n=44) * * * *
CaBP overexpression inhibits IP 3 Ester induced Calcium release M InsP 3 ester SCaBP1 Control time (s) Ca2+ i (nM) CaBP acts directly on the IP 3 R
CaBP1 inhibits IP 3 binding to the IP 3 R control sCaBP1Ca 2+ sCaBP1 100 [ 3 H]IP 3 Binding (% vs control) IP 3 binding 45 Ca 2+ flux 45 Ca 2+ -release (%) [IP 3 ] (µM) control sCaBP ,
EF1EF2EF3EF Time (s) 0 Ca 2+ i (nM) µM ATP Control CaBP 134 CaBP1 inhibits IICR independent of Ca 2+ binding (1) % responsive cells ATP ( M)
CaBP1 is not a Ca 2+ sensor for the IP 3 R 200 nM IP 3 CaBP1 134 CaBP1 [Ca 2+ ] (µM) 45 Ca 2+ flux (%) CaBP1 inhibits IICR independent of Ca 2+ binding (2)
IICR is inhibited by CaBP1 CaBP1 binds to the receptor in a Ca 2+ independent manner resulting in inhibition of IP 3 binding to its receptor. To summarise.. But, how is CaBP1 activity regulated? CaBP does not activate Ca 2+ release as previously reported
sCaBP1sCaBP1-G2A lCaBP1-G2AlCaBP1 Membrane Targeting of CaBP may regulate its activity (1) EF1EF2EF3EF4 CaBP1-G2A
ATP (µM ) Membrane Targeting of CaBP1 may regulate its activity (2) time (s) Control sCaBP -G2A Ca 2+ i (nM) % responsive cells ATP ( M) Peak amplitude (nM) * * * *
32 P- CaBP YFPS120A wt Time (s) Ca 2+ i (nM) ATP (µM) 100 Control CaBP1 S120A ATP ( M) Peak amplitude (nM) * * % responsive cells * * * Phosphorylation of CaBP1 decreases it’s potency EF1EF2EF3EF4 S120A CaBP1 S120A Casein kinase [ST]xx[DE]
10µM CCh 0.5mM Caf 1mM Caf 2mM Caf [ C a 2 + ] i ( n M ) CaBP1 does not Affect Ca 2+ release through RyRs Time (s) Control sCaBP1
Summary CaBP inhibits IICR CaBP binds to the InsP 3 R inhibiting IP 3 binding CaBP activity is Calcium independent Myristoylation is not necessary for CaBP activity, although it may have a regulatory role Phosphorylation of CaBP decreases it’s potency RyRs are not modulated by CaBP In Neurons: CaM preferentially modulates RyRs CaBPs preferentially modulate IP 3 R?
CaBP1CaM Neurons IP 3 RRyR In Neurons: CaM preferentially modulates RyRs CaBPs preferentially modulate IP 3 R? Conclusions
VGCC LGCC SOCC (TRP) MSCC PMCA NCX IP 3 RRYR ER/SR SERCA IP 3 R Golgi PMR1 Mitochondria NCX Uniporter Others?? Buffers Second messenger
Control Calbindin 0.5µM ATP1µM ATP100µM ATP Time (s) Ca2+ i (nM) Inhibition of IICR by CaBP is not due to Calcium Buffering
% responsive cells M1M1M0.5 M 0 Peak Amplitude (nM) ATP Control Calbindin Calbindin Decreases Peak Response without decreasing number of responsive cells