Increased Ca 2R Storage Capacity of the Skeletal Muscle Sarcoplasmic Reticulum of Transgenic Mice Over-Expressing Membrane Bound Calcium Binding Protein Junctate ALEXANDRA DIVET, 1 SILVIA PAESANTE, 1 CRISTIANO GRASSO, 1 DARIO CAVAGNA, 1 CECILIA TIVERON, 2 CECILIA PAOLINI, 3 FELICIANO PROTASI, 3 CORINNE HUCHET-CADIOU, 4 SUSAN TREVES, 5 AND FRANCESCO ZORZATO 1,5 * 1 Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Ferrara, Italy 2 Transgenic Mice Service Center, Experimental Research Center, Regina Elena Institute, Roma, Italy 3 Interuniversitary Institute of Myology, CeSI, Center for Research on Aging, University G. d’Annunzio School of Medicine, Chieti, Italy 4 UMR CNRS U3B 6204, Faculty of Sciences and Techniques, University of Nantes, Nantes, France 5 Department of Anesthesia and Research, Basel University Hospital, Basel, Switzerland Junctate is an integral sarco(endo)plasmic reticulum protein expressed in many tissues including heart and skeletal muscle. Because of its localization and biochemical characteristics, junctate is deemed to participate in the regulation of the intracellular Ca 2þ concentration. However, its physiological function in muscle cells has not been investigated yet. In this study we examined the effects of junctate over-expression by generating a transgenic mouse model which over-expresses junctate in skeletal muscle. Our results demonstrate that junctate over-expression induced a significant increase in SR Ca 2þ storage capacity which was paralleled by an increased 4-chloro-m- cresol and caffeine-induced Ca 2þ release, whereas it did not affect SR Ca 2þ -dependent ATPase activity and SR Ca 2þ loading rates. In addition, junctate over-expression did not affect the expression levels of SR Ca 2þ binding proteins such as calsequestrin, calreticulin and sarcalumenin. These findings suggest that junctate over-expression is associated with an increase in the SR Ca 2þ storage capacity and releasable Ca 2þ content and support a physiological role for junctate in intracellular Ca 2þ homeostasis. J. Cell. Physiol. 213: 464–474, 2007. ß 2007 Wiley-Liss, Inc. Alterations of the intracellular calcium concentration, [Ca 2þ ] i , are involved in numerous cellular functions in excitable as well as non-excitable cells. In many cells, the interaction of ligands with specific receptors present on the plasma membrane coupled to PIP2 hydrolysis and IP3 generation, leads to a rapid but transient increase in the cytoplasmic [Ca 2þ ]. In view of its role as a second messenger, the free cytosolic [Ca 2þ ] cannot remain elevated for a long period of time and its concentration is tightly regulated by many proteins and organelles. In striated muscles, the sarcoplasmic reticulum (SR) which is a highly specialized form of the endoplasmic reticulum (ER), represents the main intracellular Ca 2þ storage compartment and plays a key role in excitation–contraction (EC) coupling (for reviews see Melzer et al., 1995; Berchtold et al., 2000). In skeletal muscle, electrical depolarization of the plasma membrane leads to an increase in the myoplasmic [Ca 2þ ] which in turn activates the contractile apparatus, leading to muscle contraction, a phenomenon known as EC coupling. The anatomical site of EC coupling is the calcium release unit (CRU), a unique intracellular synapse made up by two membrane compartments the extracellular sarcolemma and transverse (T) tubular system, and the sarcoplasmic reticulum (SR) terminal cisternae. The transverse (T) tubular system is an invagination of the sarcolemma into the muscle fibers and its lumen communicates with the extracellular space. CRUs may be formed by the direct association of the SR with the plasma membrane (peripheral couplings, mostly found in developing muscles) or by the association of three elements, a central transverse T tubule, and two closely apposed terminal cisternae of the SR (triad, representing mature CRUs). Two macromolecular complexes containing two distinct Ca 2þ channels are located on the triad and are intimately involved in EC coupling: Abbreviations: 4-CmC, 4-chloro-m-cresol; CRU, calcium release unit; DHPR, dihydropyridine receptors; EC, excitation– contraction; EDL, extensor digitorum longus; EM, electron microscopy; ER, endoplasmic reticulum; HRC, histidine-rich Ca 2þ binding; IP3R, inositol triphosphate receptors; JFM, junctional face membrane; PCR, polymerase chain reaction; RyR, ryanodine receptors; SR, sarcoplasmic reticulum; TRPC, transient receptor potential channels; T tubule, transverse tubule; TG, junctate transgenic mouse; WT, wild type mouse. Contract grant sponsor: European Union; Contract grant number: HPRN-CT-2002-00331. Contract grant sponsor: Italian Telethon Foundation; Contract grant number: GGP030289. *Correspondence to: Francesco Zorzato, Dipartimento di Medicina Sperimentale e Diagnostica, Sez. Patologia Generale, Universita ` degli Studi di Ferrara, Via Borsari 46, 44100 Ferrara, Italy. E-mail: zor@unife.it or fzorzato@uhbs.ch Received 22 January 2007; Accepted 28 March 2007 DOI: 10.1002/jcp.21121 ORIGINAL ARTICLE 464 Journal of Journal of Cellular Physiology Cellular Physiology ß 2007 WILEY-LISS, INC.