Abstract
Triadin in skeletal muscle exists as a disulfide linked oligomer. Triadin is solubilized in CHAPS after reduction to its monomer. Purified reduced triadin is not retained on a hydroxylapatite (HAPT) column in the presence of 30 mM potassium phosphate (KP$\sb{\rm i}$), while the junctional foot protein (JFP) and dihydropyridine receptor (DHPr) purified in the absence of triadin are retained. In contrast, triadin solubilized as a detergent extract of reduced triadic vesicles is retained by the HAPT column and elutes concomitantly with the JFP and DHPr.Triadin derived from a detergent extract of reduced vesicles is retained by HAPT in the presence of 180 mM KP$\sb{\rm i}$ which elutes a portion of the JFP and DHPr. Triadin elutes with the remaining portion of JFP and DHPr upon the addition of KCl (820 mM) to the 180 mM KP$\sb{\rm i}$ medium. Several lines of evidence support the existence of a complex between triadin, JFP and DHPr in the high salt extract: (1) All three proteins co-elute in the void volume of molecular sieve column; (2) a portion of triadin co-migrates with the DHPr but separates from the JFP upon rate zonal centrifugation; and (3) the complex is immunoprecipitated by monoclonal antibodies directed against the individual proteins. These results demonstrate a role for triadin as the linkage in forming a ternary complex between the JFP and DHPr at the triad junction.$\lbrack\sp{125}$I$\rbrack$JFP binds to triadin in a saturable manner. The binding becomes weaker in hypertonic salt concentrations. Autoradiography of the overlays confirm that binding still occurs even in 0.5 M salt. Scatchard analysis determined that $\lbrack\sp{125}$I$\rbrack$JFP bound to triadin with a B$\sb{\rm max}$ of 100 pmol mg$\sp{-1}$ and a K$\sb{\rm d}$ of 40 nM.The influence of various ligands on binding was investigated using $\lbrack\sp{125}$I) JFP and triadin immobilized on nitrocellulose. Ca$\sp{2+}$ inhibits binding whereas, Mg$\sp{2+}$ produces a dose dependent increase in binding. Furthermore, ATP and ruthenium red inhibits binding. These data indicate that the potency of binding between triadin and the JFP is controlled in a complex manner by activators or inhibitors of channel opening.