Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -Binding Proteins Laboratory of Physiology...

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Transcript of Regulation of the IP 3 Receptor by Ca 2+ and Ca 2+ -Binding Proteins Laboratory of Physiology...

  • Regulation of the IP3 Receptor by Ca2+ and Ca2+-Binding ProteinsLaboratory of PhysiologyKULeuvenLeuven, Belgium

  • VGCCLGCCSOCCMSCCPMCANCXIP3RRYRER/SRSERCAIP3RgolgiPMR1MitochondriaNCXUniporterOthers??BuffersRSecond messenger

  • IP3RI, II, III

  • Ca2+ is the primary modulator of its own release by intracellular Ca2+ release channelsStructure of the IP3RRegulation of the IP3R by Ca2+Bell-shaped IICR

  • Effect of Calmodulin on IP3-induced Ca2+ releaseA7r5HBE

    Chart8

    0.700.3

    0.050.050.9

    R1

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    R3

    Sheet1

    R1Ca2+NOCaM

    0.0013032

    0.034746

    0.15555

    0.35621

    0.62010

    R30.0011719

    0.031822

    0.12115

    0.3205

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    192

    R10.70.05

    R200.05

    R30.30.9

    Sheet1

    0.001 0.03 0.1 0.3 0.6 1

    0.001 0.03 0.1 0.3 0.6 1

    [Ca2+] (M)

    Ca2+ release (% / 2min)

    Sheet2

    [Ca2+] (M)

    Ca2+ release (% / 2min)

    Sheet3

    R1

    R2

    R3

    IP3R expression

    R1

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  • Calmodulin and apocalmodulinSienaert, 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 receptorBiochem. J. 365, 269-277, 2002

  • Lbs-1 Lbs-1 1-2251581W226581NH2COOHERCYTLbs-1: IP3 binding core (226-604)CaM Ca2+CaM Recombinant ligand-binding domain of IP3R1 (LBS-1) 1-225(Adkins et al., 2000)

  • Calmodulin effect on IP3 bindingCaM inhibits IP3 binding in Ca2+ -independent way

  • Localisation of a Calmodulin-Binding Site

  • 1- B/BoDetailed localisation using peptidesCa2+ independent CaM-binding site in the N-terminal Region

  • Calmodulin binding sites on IP3R1Ca2+/CaM

  • 1000750500250060040020000.51100Ca2+i (nM)ControlIICR is inhibited by CaM and CaM1234

  • Regulation of Calcium release 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. BootmanIn preparation

  • CaM or other CaM-like Ca2+ sensor proteins ?

  • CaBP binds to the InsP3R

  • CaBP1/11/21/41/51/61/81/101/3CaBPRatio of CaBP: peptide B+ Calcium+ EGTABinding of CaBP to the InsP3R is calcium independent

  • CaBP inhibits InsP3 induced Calcium release independent of Calcium binding

  • CaBP overexpression inhibits InsP3 Ester induced Calcium release:CaBP acts directly on the InsP3R20015010012001000800600400200010 mM InsP3 ester050250Controltime (s)Ca2+i (nM)

  • SummaryCaBP inhibits IICRCaBP inhibits IP3 bindingCaBP activity is Calcium independentCaBP binds on the N-terminal CaM-binding site in a Ca2+-independent way

  • Suramin Interacts with the CaM-binding sites on the IP3R1

  • MLCK peptide inhibits IICRHigh CaM affinity binding properties

  • Inhibition of IICR by other CaM-binding peptides?Properties of the inhibition of IICR by MLCK peptide

  • SummarySuramin Inhibits IICR by lowering the IC50 for activation by IP3MLCK peptide inhibits IICR by lowering Vmax Suramin interacts with the CaM-binding sites on the IP3RBy removing endogenous CaM?By disrupting intermolecular protein-protein interaction?

  • ConclusionsTwo CaM-binding sites on the IP3R Ca2+ dependent in the regulatory domainCa2+ independent in the N-terminus CaM inhibts IICR only in the presence of Ca2+ Ca2+ is the major regulator of the IP3R 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 cellsN. 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 cellsFractional loss

  • Ca2+ -induced Ca2+ Release in A7r5 cells

  • 01020010203040Time (min)Effects of CaM, CaM1 and CaM1234controlcontrolCaMCaM1CaM1234

  • Calmodulin1CalmodulinCalmodulinCalmodulin1234Long CaBP1Short CaBP1NCS-1/FrequeninE120QCalmodulin1234Long CaBP1NCS-1/FrequeninNCS-1/FrequeninShort CaBP1NCS-1/FrequeninE120QCalmodulin1

  • Ca2+ (3 M)controlRyR CaM-BS (peptide aa 3614-3643)Preincubation with a CaM-binding peptide inhibits CICR

  • CaM but not CaM1234 can restore CICRPreincubation with RyR CaM-BS (peptide aa 3614-3643)Ca2+ (3 M)

  • CaM but not CaM1234 can restore CICRPreincubation with RyR CaM-BS (peptide aa 3614-3643)Ca2+ (3 M)CaM

  • SummaryNovel CICR mechanism in A7r5 cells CICR mechanism is regulated by CaMIC50: 700 nMActivated by ATPInhibited by MgCl2

  • 27492275224576G

  • Lab. of Physiology K.U.L., BelgiumBrabaham Institute, Cambridge, UKLlewelyn RoderickMartin BootmanAndreas Jeromin Baylor College of Medicine, Houston, Texas