6130 Biochemistry 2010, 49, 6130–6135 DOI: 10.1021/bi100796v Caveolin 3 Is Associated with the Calcium Release Complex and Is Modified via in Vivo Triadin Modification† Stephane Vassilopoulos,‡,§,@ Sarah Oddoux,‡,§,@ Severine Groh, )Marine Cacheux,‡,§ Julien Faure,‡,§,^ Julie Brocard,‡,§ Kevin P. Campbell, )and Isabelle Marty*,‡,§,^ ‡INSERM U836, Grenoble Institut des Neurosciences, Equipe Muscle et Pathologies, Grenoble, France, §Universite Joseph Fourier, Grenoble, France, H)oward Hughes Medical Institute and Departments of Molecular Physiology and Biophysics, Neurology, and Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242, and ^CHRU de Grenoble, Hopital Michallon, Biochimie et Genetique Moleculaire, Grenoble, France. @These authors contributed equally to this work. Received May 19, 2010; Revised Manuscript Received June 18, 2010 ABSTRACT: The triadin isoforms Trisk 95 and Trisk 51 are both components of the skeletal muscle calcium release complex. To investigate the specific role of Trisk 95 and Trisk 51 isoforms in muscle physiology, we overexpressed Trisk 95 or Trisk 51 using adenovirus-mediated gene transfer in skeletal muscle of newborn mice. Overexpression of either Trisk 95 or Trisk 51 alters the muscle fiber morphology, while leaving un- changed the expression of the ryanodine receptor, the dihydropyridine receptor, and calsequestrin. We also observe an aberrant expression of caveolin 3 in both Trisk 95- and Trisk 51-overexpressing skeletal muscles. Using a biochemical approach, we demonstrate that caveolin 3 is associated with the calcium release complex in skeletal muscle. Taking advantage of muscle and non-muscle cell culture models and triadin mouse skeletal muscle, we further dissect the molecular organization of the caveolin 3-containing calcium release complex. Our data demonstrate that the association of caveolin 3 with the calcium release complex occurs via a direct interaction with the transmembrane domain of the ryanodine receptor. Taken together, these data suggest that caveolin 3-containing membrane domains and the calcium release complex are functionally linked and that Trisk 95 and Trisk 51 are instrumental to the regulation of this interaction, the integrity of which may be crucial for muscle physiology. In skeletal muscle, the excitation-contraction (EC)1 coupling process takes place at the triads where T-tubules and the re- ticulum sarcoplasmic terminal cisternae are in close contact. EC coupling requires the expression at the triads of a multimeric calc- ium release complex (CRC) that includes the T-tubule voltage- dependent calcium channel dihydropyridine receptor (DHPR) and several sarcoplasmic proteins, namely, the calcium release channel ryanodine receptor (RyR) (1, 2), the sarcoluminal Ca2þ binding calsequestrin (CSQ), and two of the four triadin isoforms identified to date in skeletal muscle, Trisk 95 (T95) and Trisk 51 (T51) (3, 4). In vitro studies had identified a couple of regulatory RyR binding domains on triadin, and these regions are common to both Trisk 95 and Trisk 51 (5, 6). Adenoviral-mediated over- expression of either Trisk 95 or Trisk 51 in primary cultures of skeletal muscle further demonstrated that only Trisk 95 plays a role in the regulation of the depolarization-induced calcium †This work was supported by grants from Association Franc-aise contre les Myopathies (AFM), from Groupement d’Int^eret Scientifique (GIS)-Maladies Rares, from Agence Nationale de la Recherche (ANR- Maladies Rares), from Societe Franc-aise de Myologie (SFM), and from Fondation Ducoin. *To whom correspondence should be addressed: Grenoble Institut des Neurosciences-INSERM U836, Eq Muscle et Pathologies, Batiment EJ Safra, Chemin Fortune Ferrini, 38700 La Tronche, France. E-mail: isabelle.marty@ujf-grenoble.fr. Telephone: (33) 4 56 52 05 71. Fax: 33 4 56 52 05 72. 1Abbreviations: Cav-3, caveolin 3; CRC, calcium release complex; CSQ, calsequestrin; DHPR, dihydropyridine receptor; EC, excitation- contraction; KO, knockout; RyR, ryanodine receptor; SERCA, sarco- endoplasmic reticulum Ca2þ-ATPase; T95, Trisk 95; T51, Trisk 51; WT, wild type. release mechanism (4), suggesting a specific role of Trisk 95 in EC coupling. Interestingly, triadin mice exhibit a structural myo- pathy (7) with impaired depolarization-induced calcium release (7, 8) indicating that the triadins are likely to be involved in the development of human myopathies for which a causative gene has not yet been identified. However, no modification has been yet identified in human triadins, both Trisk 95 and Trisk 51 being expressed in human skeletal muscle (9), and it is currently unknown whether overexpression of Trisk 95 and Trisk 51 would be detrimental for muscle function in vivo. In this study, we use adenovirus-mediated gene transfer to overexpress Trisk 95 or Trisk 51 in mouse skeletal muscle as an alternative approach to investigating the function of these triadin isoforms. Herein, we show that overexpression of either Trisk 95 or Trisk 51 alters the muscle fiber morphology while leaving the expression of RyR, DHPR, and CSQ unchanged. We also observe that caveolin 3 (Cav-3), an essential structural component of caveolae involved in endocytosis and intracellular trafficking events (10), is aberrantly expressed in both Trisk 95- and Trisk 51- overexpressing skeletal muscles. We further demonstrate that Cav-3 is associated with the CRC via a direct interaction with the RyR, and we propose that the Trisk 95 and Trisk 51 level of expression is critical for the regulation of this interaction. MATERIALS AND METHODS Animals. Wild-type mice (C57BL/6) were bred at the Univer- sity of Iowa from stocks originally obtained from Jackson Labo- ratories (Bar Harbor, ME). Triadin mice have been described pubs.acs.org/Biochemistry Published on Web 06/21/2010 r 2010 American Chemical Society