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N. Nadif Kasri, I. Sienaert, S. Vanlingen, J.B. Parys, G. Callewaert, L. Missiaen, H. De Smedt

Localisation and Function of a Calmodulin/Apocalmodulin Binding Site in the N-terminal Part of the Inositol 1,4,5-Trisphosphate Receptor. N. Nadif Kasri, I. Sienaert, S. Vanlingen, J.B. Parys, G. Callewaert, L. Missiaen, H. De Smedt. Cytosol. CaM ??. 31. 25. 13. 18.

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N. Nadif Kasri, I. Sienaert, S. Vanlingen, J.B. Parys, G. Callewaert, L. Missiaen, H. De Smedt

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  1. Localisation and Function of a Calmodulin/Apocalmodulin Binding Site in the N-terminal Part of the Inositol 1,4,5-Trisphosphate Receptor. N. Nadif Kasri, I. Sienaert, S. Vanlingen, J.B. Parys, G. Callewaert, L. Missiaen, H. De Smedt

  2. Cytosol CaM ?? 31 25 13 18 Endoplasmic reticulum Functional Domains on IP3R1

  3. Effect of Ca2+ and Calmodulin on IP3-induced Ca2+ Release in A7r5 Smooth Muscle Cells • CaM • +CaM 60 release (% / 2min) 40 6x His-tag 581 20 Ca2+ 2+ Ca N N 0 IP3 CaM? C 0.6 0.3 <0.001 0.03 0.1 2+ Free [Ca ] (µM) Recombinant ligand-binding domain of IP3R1 (Lbs-1His)

  4. Functional data: Effect of Calmodulin/Apocalmodulin on 3[H]IP3 Binding to Lbs-1His Lbs-1His Lbs-1His 1-225 581 1 Constructs: Lbs-1His W226 581 Lbs-1 1-225His [3H]IP3 binding (%) control 5 µM Ca2+ Ca2+ CaM Ca2+ CaM1234 10 µM apoCaM 10 µM CaM1234

  5. 1 581 GST-fusion protein pull down of CaM1234 Lbs-1 W226 581 Lbs-1 1-225 pGST GST-Cyt1 GST-Cyt2 309 159 1 50 M free Ca2+ Cyt1 Cyt2 CaM1234 1 mM EGTA CaM1234 Localisation of a Calmodulin-Binding Site

  6. 40 nM Dan-CaM EGTA 40 nM Dan-CaM EGTA 40 nM Dan-CaM + 0.1 µM GST-Cyt1 + 0.1 µM pGST + 0.5 mM Ca2+ + 0.1 µM GST-Cyt1 Dansyl-Calmodulin Experiments Excitation at 340 nm Emission max. at 500 nm Relative Fluorescence Wavelength (nm)

  7. 159 1 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ (site1) A C D E B F F/F0500 Detailed localisation using peptides

  8. Dansyl-Calmodulin Experiments Peptide E: aa 106-128 40 nM Dan-CaM 1 mM EGTA + 1 µM peptide E 1 mM EGTA 50 µM free Ca2+ Relative Fluorescence Fraction Dan-CaM bound Kd 1µM [Peptide E] (µM) Wavelength (nm)

  9. Conclusions 1. Functional: Effect of calmodulin on [3H]IP3 binding to Lbs-1His is calcium independent. 2. Localisation of a Ca2+-independent calmodulin binding site in the N-terminal part of the IP3R1. Ca2+-independent calmodulin binding site: aa 106-128 Kd: 1µM 3. Ca2+-independent calmodulin binding site is probably responsible for the inhibition on [3H]IP3 binding.

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