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J. Biol. Chem., Vol. 281, Issue 13, 8332-8338, March 31, 2006

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Endogenously Bound Calmodulin Is Essential for the Function of the Inositol 1,4,5-Trisphosphate Receptor^*

Nael Nadif Kasri¹, Katalin Török, Antony Galione^¶, Clive Garnham^¶, Geert Callewaert, Ludwig Missiaen, Jan B. Parys, and Humbert De Smedt²

From the Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, O/N Herestraat 49/802, B-3000 Leuven, Belgium, ^¶Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom, and Department of Basic Medical Sciences, St. George's University of London, Cranmer Terrace, London SW17 0RE, United Kingdom

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays an important role in regulating a large number of Ca²⁺ channels, including the inositol 1,4,5-trisphosphate receptor (IP₃R). Despite many efforts, the exact mechanism by which CaM regulates the IP₃R still remains elusive. Here we show, using unidirectional ⁴⁵Ca²⁺ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP₃R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP₃-induced Ca²⁺ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP₃. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP₃-induced Ca²⁺ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM₁₂₃₄, reactivated the IP₃R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP₃R, and this resulted in a complete loss of the IP₃R activity. Our data support a new model whereby CaM is constitutively associated with the IP₃R and functions as an essential subunit for proper functioning of the IP₃R.

Received for publication, October 17, 2005 , and in revised form, December 19, 2005.

^* This work was supported by Grants G.0210.03 (to H. D. S. and J. B. P.) G.O0382.05 (to L. M. and G. C.) of the Fund for Scientific Research Flanders (Belgium), by Grant GOA2004/07 from the Concerted Actions of the K. U. Leuven Institute (to L. M., H. D. S., G. C., and J. B. P.) and by the Interuniversity Poles of Attraction Programme— Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs, IUAP P5/05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A postdoctoral fellow of the Research Foundation (FWO-Vlaanderen) Brussels, Belgium.

² To whom correspondence should be addressed: Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Herestraat 49/802, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-25; Fax: 32-16-34-59-91; E-mail: humbert.desmedt{at}med.kuleuven.be.

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