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Regulation of Calcium release By neuronal Ca lcium B inding P roteins ( CaBP )

Regulation of Calcium release By neuronal Ca lcium B inding P roteins ( CaBP ). H. Llewelyn Roderick. SOCE. Bezprozvanny et al., 1991. John et al., 1998. J Timofeeva & S Coombes. 12 Stimuli. 5 Stimuli. Finch and Augustine, 1998. BCl2. CnA. FKBP12. P. P. 4.1N. CRT/CLNX. 224.

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Regulation of Calcium release By neuronal Ca lcium B inding P roteins ( CaBP )

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  1. Regulation of Calcium release By neuronal Calcium Binding Proteins (CaBP) H. Llewelyn Roderick

  2. SOCE

  3. Bezprozvanny et al., 1991

  4. John et al., 1998 J Timofeeva & S Coombes

  5. 12 Stimuli 5 Stimuli Finch and Augustine, 1998

  6. BCl2 CnA FKBP12 P P 4.1N CRT/CLNX 224 576 2749 2275

  7. CaM CaM CaM-like EF1 EF2 EF3 EF4 L-CaBP1 S-CaBP1 CaBP1 Caldendrin EF1 EF2 EF3 EF4 S-CaBP5 S-CaBP2 CaBP2 EF1 EF2 EF3 EF4 L-CaBP2 S-CaBP1 CaBP3 EF3 EF4 CaBP3 GCAP GCAP3 GCAP2 EF1 EF2 EF3 EF4 Recoverin CaBP4 Visinin Neurocalcin EF1 EF2 EF3 EF4 VILIP3 CaBP5 VILIP1 VILIP2 NCS1 Adapted from Haeseleer et al., 2000

  8. Exocytosis in Synapses Exocytosis in Endocrine cells Basal Ca2+ Channel regulation 100 80 NCS1 VILIP-1 CaM Synaptotagmin 60 Ca2+ bound 40 20 0 7 6 5 4 3 -log[Ca2]+ Why so many CaBPs….? Cellular Localisation and…. Adapted from Burgoyne and Weiss 2001

  9. Adapted from Haeseleer et al., 2000

  10. EF1 EF2 EF3 EF4 CaBP1 SCaBP1 LCaBP1

  11. SCaBP LCaBP Both Long and Short CaBPs inhibit InsP3 induced Calcium release 100µM 1µM 0.5µM ATP 1000 800 600 Ca2+i (nM) 400 Control 200 0 0 200 400 600 800 1000 Time (s)

  12. SCaBP1 (n=22) LCaBP1 (n=17) Control (n=44)

  13. SCaBP1 CaBP overexpression inhibits InsP3 Ester induced Calcium release: CaBP acts directly on the InsP3R 10 mM InsP3 ester 250 200 Control 150 Ca2+i (nM) 100 50 0 0 200 400 600 800 1000 1200 time (s)

  14. CaBP inhibits InsP3 binding to the InsP3R 4 µg Lbs-1 10 µM sCaBP1 5 µM Ca2+ 100% 100 78 ± 1.9 % 68 ± 6.9 % 75 [3H]IP3 Binding (% vs control) 39 ± 4.7% 50 25 0 sCaBP1 Ca Ca/ sCaBP1 Lbs-1

  15. EF1 EF2 EF3 EF4 0.5 1 100 µM ATP 100 1000 800 % responsive cells 50 Control 600 CaBP134 Ca2+i (nM) 400 0 0.5 1 100 200 ATP (mM) 0 0 1500 500 1000 Time (s) CaBP inhibits InsP3 induced Calcium release independent of Calcium binding

  16. Calbindin Inhibition of InsP3 Induced Calcium Release by CaBP is not due to Calcium Buffering 0.5µM ATP 800 1µM ATP 100µM ATP 600 Ca2+i (nM) 400 200 Control 0 0 1500 500 1000 Time (s)

  17. Calbindin Decreases Peak Response without decreasing number of responsive cells 500 400 300 Peak Amplitude (nM) 200 100 0 0.5mM 1mM 100mM 100 % responsive cells 50 Control 0 Calbindin ATP

  18. GST 1-604 GST 1-225 GST 226-604 GST CaBP LCaBP-YFP SCaBP-YFP sCaBP134 Control YFP InsP3RIII CaBP binds to the InsP3R

  19. CaBP binds to a similar region of the InsP3R as CaM A) 159 CaM CaM 1 A C D E B F B) -Ca2+/EGTA + Ca2+ sCaBP1 C D F A B E C D F A B E sCaBP1

  20. 1.0 Band intensity 0.5 0.0 0 2 4 6 8 10 Peptide B: CaBP 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

  21. CaM CaM 1234 Control CaM IICR is inhibited by CaM 0.5 1 100 ATP (µM ) 600 Control 400 Ca2+i (nM) 200 0 0 250 500 750 1000

  22. To summarise.. • IICR is inhibited by CaBP • CaBP binds to the receptor in a calcium independent manner resulting in inhibition of IP3 binding to its receptor. CaBP does not activate calcium release as previously reported But, how is CaBP activity regulated?

  23. SCaBP1-G2A LCaBP1-G2A SCaBP1 LCaBP1

  24. 100 % responsive cells 50 0 sCaBP -G2A 900 700 500 Peak amplitude (nM) 300 100 0 ATP (mM) 0.5 1 100 Membrane Targeting of CaBP may regulate its activity 1 100 0.5 ATP (µM ) 1000 750 Control Ca2+i (nM) 500 250 0 1500 1000 0 500 time (s)

  25. StsE (S120~S101 in CaM)

  26. 100 0.5 1 100 % responsive cells 50 0 Control CaBP DCK2 900 700 Peak amplitude (nM) 500 300 1500 0 1000 500 100 0 0.5 1 100 ATP (mM) Phosphorylation of Serine 120 regulates CaBP activity ATP (µM) 1200 Ca2+i (nM) 800 400 0 time (s)

  27. 100 % responsive cells 50 0 500 Calmyrin 400 300 Peak Amplitude (nM) 200 100 0 0.5 1 100 ATP mM Calmyrin Inhibits IICR ATP (µM) 0.5 1 100 700 500 Ca2+i (nM) Control 300 100 0 0 1500 500 1000 time (s)

  28. CaBP does not Affect Ca2+ release through RyR I ) M n ( i ] + 2 a C [ sCaBP1 10µM CCh 1mM Caf 300 0.5mM Caf 2mM Caf 200 100 Control 0 0 500 1000 1500 2000 Time (s)

  29. ) M n ( i ] + 2 a C [ sCaBP 1 CaBP does not Affect Ca2+ release through RyR III 1mM Caf 10µM CCh 300 0.5mM Caf 2mM Caf 200 100 Control 0 0 500 1000 1500 2000 Time (s)

  30. Summary • CaBP inhibits IICR • CaBP binds to the InsP3R inhibiting IP3 binding • CaBP activity is Calcium independent • Myristoylation is not necessary for CaBP activity, although it may have a regulatory role. • Phosphorylation of CaBP increases it’s potency. • RyRs are not modulated by CaBP

  31. Conclusion • CaBPs may bind in preference to CaM to the InsP3R in neuronal cells regulating Calcium release. • The regulation of CaBPs by Casein Kinase II resulting in their decreased affinity for the InsP3R and resulting increased InsP3R sensitivity provides a mechanism by which LTP may occur.

  32. Ongoing/Future Work • Express CaBP in primary neuronal culture • Express CaBP in Brain slices and examine LTP/LTD • Inject CaBP protein into Xenopus oocytes and investigate effect on calcium puffs

  33. Acknowledgments Babraham Institute Laboratorium voor Fysiologie, K.U. Leuven Martin Bootman Michael Berridge Nael Nadif Kasri Geert Bultynck Jan B Parys Ludwig Missiaen Humbert De Smedt Anthony Holmes Tony Collins Lauren Mackenzie Jane Hanson Paul Cuddon Andrew Proven Claire Peppiatt Rod O’Connor Steve Maddison Brynn Kvinlaug Clark Distelhorst

  34. Diolch Yn Fawr

  35. CCE is enhanced by InsP3. Cardiac function is Modulated by InsP3/InsP3Rs CCE is regulated by interactions between InsP3Rs and TRP channels. InsP3Rs are modulated by Protein-protein interactions e.g. neuronal CaBPs. A multiple role for InsP3Rs in cell signalling InsP3.R

  36. 0.5µM ATP 1µM ATP 100µM ATP 800 600 400 200 0

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