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J. Biol. Chem., Vol. 280, Issue 15, 15380-15389, April 15, 2005

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Ca²⁺ Signaling in HEK-293 and Skeletal Muscle Cells Expressing Recombinant Ryanodine Receptors Harboring Malignant Hyperthermia and Central Core Disease Mutations^*

Marisa Brini¶**, Sabrina Manni**, Nicola Pierobon, Guo Guang Du||, Parveen Sharma||, David H. MacLennan||, and Ernesto Carafoli**

From the Department of Biochemistry and Center for the Study of Biomembranes of the National Research Council (CNR), University of Padova, Viale G. Colombo 3, 35121 Padova, Italy and the ^**Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35129 Padova, Italy, the Department of Experimental Veterinary Sciences, Viale dell'Università 16, Agripolis, Legnaro Padova, Italy, and the ^||Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca²⁺ release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca²⁺ pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca²⁺ homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca²⁺ revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca²⁺ transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca²⁺ derangements of different degrees account for the specific cellular phenotypes of the two disorders.

Received for publication, September 10, 2004 , and in revised form, February 2, 2005.

^* This work was supported by the Telethon Foundation of Italy (Grant GP0193Y01, to E. C.), by contributions from the Italian Ministry of University and Scientific Research (PRIN 2001, 2002, 2003; FIRB 2001; to M. B. and E. C.), by the National Research Council of Italy (Target Project on Biotechnology and Agency 2000), by the Human Frontier Science Program Organization, by Grants MT-3399 and MOP-49493 from the Canadian Institutes of Health Research, by the Neuromuscular Research Partnership (to D. H. M.), and by a postdoctoral fellowship (to P. S.) from the Heart and Stroke Foundation of Canada. 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.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, University of Padova, Viale G. Colombo, 3, 35121 Padova Italy. Tel.: 39-049-8276167; Fax: 39-049-8276125; E-mail: brini{at}mail.bio.unipd.it.

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