To identify the function of triadin in skeletal muscle, adenovirusmediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex. In skeletal muscle cells, excitation-contraction (E-C)2 coupling is the process by which depolarization of the plasma membrane produces a large transient release of Ca 2ϩ from the sarcoplasmic reticulum, which in turn triggers contraction. Dihydropyridine receptors (DHPRs), the L-type Ca 2ϩ channels localized in the T-tubule membrane, serve as the voltage sensors for E-C coupling (1, 2) and activate ryanodine receptors (RyRs), the intracellular Ca 2ϩ release channels of the sarcoplasmic reticulum membrane (3, 4). In skeletal muscle, entry of Ca 2ϩ through DHPR is not required for E-C coupling (5). Instead, a voltage-dependent conformational change in the II-III loop of the skeletal muscle DHPR ␣1 subunit (Cav1.1) activates the skeletal muscle ryanodine receptor, RyR1. Activation of RyR1 results in a release of Ca 2ϩ from the sarcoplasmic reticulum intracellular stores into the cytosol, which in turn activates the cellular contractile apparatus to initiate muscle contraction. In cardiac muscle, the entry of external calcium through DHPR is required to induce activation of RyR and release of internal Ca 2ϩ via RyR, a mechanism known as calcium-induced calcium release. Calcium-induced calcium release is not the initial mechanism responsible of Ca 2ϩ release in skeletal muscle, but it can contribute to the amplification of the signal, and both calcium-induced calcium release and conformational coupling are involved in skeletal muscle contraction, to different extents (6).Triadin is a transmem...

We have cloned two new triadin isoforms from rat skeletal muscle, Trisk 49 and Trisk 32, which were named according to their theoretical molecular masses (49 and 32 kDa, respectively). Specific antibodies directed against each protein were produced to characterize both new triadins. Both are expressed in adult rat skeletal muscle, and their expression in slow twitch muscle is lower than that in fast twitch muscle. Using double immunofluorescent labeling, the localization of these two triadins was studied in comparison to wellcharacterized proteins such as ryanodine receptor, calsequestrin, desmin, Ca 2؉ -ATPase, and titin. None of these two triadins are localized within the rat skeletal muscle triad. Both are instead found in different parts of the longitudinal sarcoplasmic reticulum. We attempted to identify partners for each isoform: neither is associated with ryanodine receptor; Trisk 49 could be associated with titin or another sarcomeric protein; and Trisk 32 could be associated with IP 3 receptor. These results open further fields of research concerning the functions of these two proteins; in particular, they could be involved in the set up and maintenance of a precise sarcoplasmic reticulum structure.Skeletal muscle cells have highly organized structures. Sarcomeres, the contractile units of striated muscles, are assembled from thousands of proteins to produce the largest and most regular macromolecular complex known. In addition, this organized structure is designed to undergo strong mechanical stress during muscle contraction. Muscle contraction is activated by Ca 2ϩ release from the sarcoplasmic reticulum in response to plasma membrane depolarization. This process is referred to as excitation-contraction coupling and takes place at the skeletal muscle triad junction, where T-tubules and the sarcoplasmic reticulum terminal cisternae are in close contact (1). To perform its function, the sarcoplasmic reticulum is built as a sleeve-like structure around the myofibrils, and it is compartmentalized in highly specialized structures (terminal cisternae and longitudinal reticulum) with specialized functions (calcium release and calcium uptake, respectively) (2).Calcium release occurs via a macromolecular complex, the calcium release complex, specifically localized in the skeletal muscle triad (3). Major components of this calcium release complex are the two calcium channels, the ryanodine receptor (RyR) 1 and the dihydropyridine receptor (4). Triadin is an integral membrane protein of the sarcoplasmic reticulum, first identified in rabbit skeletal muscle in 1990 (5, 6) as a 95-kDa glycoprotein specifically located in the triads. Because of its co-localization with RyR in the triads, involvement of triadin in excitation-contraction coupling has been presumed (7, 8). Protein interaction studies have shown that molecular partners of triadin are RyR (9 -11); calsequestrin (CSQ), the protein that traps calcium inside the sarcoplasmic reticulum (12-14); junctin (15); and histidine-rich Ca 2ϩ -binding protein (16). F...