Interaction of triadin with histidine-rich Ca 2+ -binding protein at the triadic junction in skeletal muscle ®bers ROBERTA SACCHETTO, FEDERICA TURCATO, ERNESTO DAMIANI and ALFREDO MARGRETH* Department of Experimental Biomedical Sciences, National Research Council Unit for Muscle Biology and Physiopathology, University of Padova, viale G. Colombo, 35121 Padova, Italy Received 10 March 1999; accepted in revised form 20 April 1999 Abstract The present study documents the binding interaction of skeletal muscle sarcoplasmic reticulum (SR) transmem- brane protein triadin with peripheral histidine-rich, Ca 2+ -binding protein (HCP). In addition to providing further evidence that HCP coenriches with RyR1, FKBP-12, triadin and calsequestrin (CS) in sucrose-density-puri®ed TC vesicles, using speci®c polyclonal antibody, we show it to be expressed as a single protein species, both in fast-twitch and slow-twitch ®bers, and to identically localize to the I-band. Colocalization of HCP and triadin at junctional triads is supported by the overlapping staining pattern using monoclonal antibodies to triadin. We show a speci®c binding interaction between digoxigenin-HCP and triadin, using ligand blot techniques. The importance of this ®nding is strengthened by the similarities in binding anity and in Ca 2+ dependence, (0.1±1 mM Ca 2+ ) of the interaction of digoxigenin-HCP with immobilized TC vesicles. Suggesting that triadin dually interacts with HCP and with CS, at distinct sites, we have found that triadin-CS interaction in overlays does not require the presence of Ca 2+ . Consistent with the binding of CS to triadin luminal domain (Guo and Campbell, 1995), we show that binding sites for digoxigenin-CS, although not binding sites for digoxigenin-HCP, can be recovered in the 92 kDa triadin fragment, after chymotryptic cleavage of the NH 2 -terminal end of the folded molecule in intact TC vesicles. These dierential eects form the basis for the hypothesis that HCP anchors to the junctional membrane domain of the SR, through binding to triadin short cytoplasmic domain at the NH 2 terminus. Although the function of this interaction, as such, is not well understood, it seems of potential biological interest within the more general context of the structural-functional role of triadin at the triadic junction in skeletal muscle. Abbreviations: CaM: calmodulin; CS: calsequestrin; HCP: histidine-rich, Ca 2+ -binding protein; RyR1: ryanodine receptor, skeletal isoform; SR: sarcoplasmic reticulum. Introduction In mammalian skeletal muscle ®bers, the sarcoplasmic reticulum (SR) Ca 2+ -release channel (RyR1) in its native membrane environment, i.e. the junctional mem- brane of terminal cisternae (TC), is present as a complex with FKBP-12 (Jayaraman et al., 1992), which seems to be crucial for coordinated ryanodine receptor (RyR1) gating (Marx et al., 1998), and further interacts struc- turally (Caswell et al., 1991) and functionally (Liu and Pessah, 1994) with transmembrane protein triadin (Knudson et al., 1993a; Lewis Carl et al., 1995). This binding interaction, like the interaction of triadin with compartmental Ca 2+ -binding protein calsequestrin (CS), was localized to triadin large cytoplasmic domain (Guo and Campbell, 1995). Triadin cytoplasmic domain, comprising only 47 amino acids from the NH 2 -terminal end of the protein, according to the same model (Knudson et al., 1993b), would be too short to reach the DHPR across the junctional gap between TC and transverse tubules (TT) (Knudson et al., 1993a, 1993b; Marty et al., 1995). That seems to dispute the early hypothesis that triadin might be the intermediate protein in electro-mechanical coupling between dihydropyridine receptors (DHPRs) and RyRs (Brandt et al., 1992). Neither it seems to be necessary for triad formation, i.e. for the docking of junctional TT membrane to the corresponding membrane domain of SR TC (see Fluc- her and Franzini-Armstrong, 1996; Sutko and Airey, 1996). This is equally supported by electron microscopic studies of muscle cells from mice homozygous for the muscular dysgenesis mutation (Franzini-Armstrong et al., 1991) and of dyspedic (RyR1 null) mouse muscle (Takekura et al., 1995). Recent evidence, using western blot techniques, has further indicated that the lack of expression of RyR1 in dyspedic muscle does not prevent the expression of triadin and of other junctional SR- speci®c proteins, such as FKBP-12 and CS (Buck et al., 1997). These several ®ndings, taken together, leave open *To whom correspondence should be addressed: Fax: (39) 49 827 6040 Journal of Muscle Research and Cell Motility 20: 403±415, 1999. 403 Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.