Interaction of triadin with histidine-rich Ca2+-binding protein at the triadic junction in skeletal muscle fibers

The present study documents the binding interaction of skeletal muscle sarc oplasmic reticulum (SR) transmembrane protein triadin with peripheral histi dine-rich, Ca2+-binding protein (HCP). In addition to providing further evi dence that HCP coenriches with RyR1, FKBP-12, triadin and calsequestrin (CS ) in sucrose-density-purified TC vesicles, using specific polyclonal antibo dy, we show it to be expressed as a single protein species, both in fast-tw itch and slow-twitch fibers, and to identically localize to the I-band. Col ocalization of HCP and triadin at junctional triads is supported by the ove rlapping staining pattern using monoclonal antibodies to triadin. We show a specific binding interaction between digoxigenin-HCP and triadin, using li gand blot techniques. The importance of this finding is strengthened by the similarities in binding affinity and in Ca2+ dependence, (0.1-1 mM Ca2+) o f the interaction of digoxigenin-HCP with immobilized TC vesicles. Suggesti ng that triadin dually interacts with HCP and with CS, at distinct sites, w e have found that triadin-CS interaction in overlays does not require the p resence of Ca2+. Consistent with the binding of CS to triadin luminal domai n (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 NH2-terminal end o f the folded molecule in intact TC vesicles. These differential effects for m the basis for the hypothesis that HCP anchors to the junctional membrane domain of the SR, through binding to triadin short cytoplasmic domain at th e NH2 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.