6134 Biochemistry, Vol. 49, No. 29, 2010 FIGURE 4: Co-immunoprecipitation of Cav-3 with the RyR. (A) Immunoprecipitation was performed on mouse skeletal muscle from a WT or triadin KO mouse with antibodies against RyR (lanes 1 and 3) or with preimmune serum (lanes 2 and 4). The immunoprecipitated proteins were then analyzed by Western blotting with antibodies against the RyR or Cav-3. (B) Immunoprecipitation was performed with anti-GFP antibodies on COS-7 cells cotransfected with GFP and Cav-3 (lane 5) or GFP-RyR and Cav-3 (lane 6). The immunopre- cipitated proteins were then analyzed by Western blotting with anti- bodies against GFP or Cav-3. experiments were performed in a non-muscle cell line. COS-7 cells were cotransfected either with Cav-3 and GFP-RyR transmem- brane domain (last 582 amino acids fused in frame with GFP on its N-terminal part) or with Cav-3 and GFP alone. The anti-GFP antibody efficiently co-immunoprecipitated the RyR and Cav-3 (Figure 4B, lane 6) but failed to co-immunoprecipitate Cav-3 in the presence of GFP alone (Figure 4B, lane 5), confirming the direct interaction between Cav-3 and the RyR1 transmembrane domain. DISCUSSION In vivo overexpression of the triadin isoform Trisk 95 or Trisk 51 was induced by adenoviral-mediated gene transfer in the hind- limb muscles of newborn mice. Overexpression of one triadin iso- form induced a reduction in the expression level of the second one. The different triadin isoforms are issued of the alternative splicing of the same gene (9), and even if the mechanisms driving the specific expression of each isoform are unknown, it is clear that the two triadins Trisk 95 and Trisk 51 not only are always associated together, as seen in the co-immunoprecipitation ex- periments, but also mutually regulate their expression, to keep the total amount of triadin in the skeletal muscle almost constant. The expression of the main components of the calcium release complex, i.e., RyR, DHPR, the Ca2þ-ATPase SERCA, and CSQ, was not affected. However, an interesting finding of our study was the alteration of Cav-3 expression in triadin-infected fibers. Using a biochemical approach, we demonstrated the ex- istence of a molecular complex, including the triadins and Cav-3. Using differential immunoprecipitations in WT and triadin skeletal muscle, as well as in muscle and non-muscle cells, we further demonstrated that Cav-3 was associated with the CRC via a direct interaction with the transmembrane domain of RyR1. Our data also suggest that two different populations of CRC exist in the skeletal muscle triads, one involving a DHPR-RyR-Cav-3 complex and one involving a Trisk 95-Trisk 51-RyR-Cav-3 complex, as shown in Figure 3. This could be related to the early electronic microscopy study demonstrating that only half of the RyR was coupled to the DHPR (1), and one could imagine that the DHPR and triadin are mutually excluding each other in the CRC, the RyR uncoupled to DHPR interacting with triadin. Triadin (Trisk 95 or Trisk 51) overexpression results in an increased level of intracellular Cav-3 staining, which is indicative of an in vivo functional link between the two proteins. As we Vassilopoulos et al. cannot demonstrate Cav-3 overexpression equivalent to triadin overexpression, this increased level of intracellular Cav-3 labeling is most probably due to Cav-3 relocalization. Our finding that Cav-3 is functionally linked to the proteins of the CRC raises the question of the physiological relevance of the association between the caveolae and the SR. Cav-3 was proposed to play a role in calcium homeostasis (21-23), and previous studies showed that the IP3R and SERCA, both involved in calcium homeostasis (24), were detected within the caveolae of several different cell types, including muscle cells (21, 22). More recently, Li et al. (24) showed the presence of microdomains enriched in cholesterol, sphingolipids, and Cav-3 in the SR membrane. Assuming that the caveolae-SR contacts are partly responsible for the control of the cytoplasmic free Ca2þ concentrations in very precisely de- fined spaces (25), the CRC involving triadins could be located at those sites. Previous studies showed that caveolae are sites of ex- tracellular Ca2þ entry (26) and suggested that caveolae are invol- ved in refilling depleted intracellular calcium stores, a mechanism called store-operated calcium entry (SOCE). Caveolae could then act as intermediates between the SR and the plasma membrane. We have previously shown that Trisk 95 can modulate the mechanism coupling the depletion of intracellular Ca2þ stores to extracellular Ca2þ entry. The overexpression of Trisk 95 dec- reases the level of this Ca2þ entry in myotubes (27). It would therefore be possible that the overexpression of Trisk 95 disturbs the SOCE by its effect on Cav-3. The relocalization of Cav-3 from the plasma membrane to intracellular compartments follow- ing the overexpression of Trisk 95 would directly disturb the Ca2þ entry by caveolae and would therefore reduce the level of store-operated Ca2þ entry. Both Cav-3 mice and transgenic mice overexpressing Cav-3 display a dystrophic phenotype (17). A likely explanation for the Cav-3 mice phenotype is the alteration of the intracellular trafficking and targeting of membrane proteins and protein complexes, such as dysferlin (2, 28) and the dystrophin glyco- protein complex (17). Noteworthy is the fact that Cav-3 skeletal muscle also exhibits abnormal DHPR and RyR staining, suggesting that Cav-3 is involved in triad formation during muscle development (29, 30). Trisk 95- and Trisk 51-overexpres- sing muscles exhibited obvious signs of pathology that included central nucleated fibers and heterogeneous fiber sizes that resemble those of dystrophic muscles. Given that Trisk 95 and Trisk 51 seem to mutually regulate their expression level and given that triadin mice exhibit muscle weakness (7), it is tempting to speculate that a precise expression level of the triadins is crucial for normal muscle function (31); an excess of triadin could be as deleterious as a lack of triadin on the muscle physiology. However, while Trisk 95 overexpression was shown to block EC coupling in skeletal muscle (4), Trisk 51 over- expression has no effect on EC coupling (4). Therefore, it is unlikely that the muscle pathology observed in triadin-over- expressing muscle results entirely from EC coupling dysfunction. In this context, one could speculate that Trisk 95 or Trisk 51 overexpression perturbs the traffic of proteins forming the Ca2þ release machinery by Cav-3 trapping, thus resulting in the ab- normal muscle phenotype observed here. A mechanism of Cav-3 trapping has been proposed for the mutant form of Cav-3, P104L, responsible for some cases of myopathy (LGMD1C) (32, 33); therefore, this mechanism has already been shown to be respon- sible for a pathology. Although no disease-causing mutation has been identified yet in the triadin gene, it is possible that modification of the triadin expression level underlies a new pathophysiological