EXTRACTION OF JUNCTIONAL COMPLEXES FROM TRIAD JUNCTIONS OF RABBIT SKELETAL-MUSCLE

Triadin in skeletal muscle exists as a disulfide linked oligomer. It d oes not dissolve well in CHAPS detergent even in 1 M KCl, but is solub ilized after reduction to its monomer by the addition of 2-mercaptoeth anol. Purified reduced triadin is not retained on a hydroxylapatite co lumn in the presence of 30 mM Potassium phosphate, while the junctiona l foot protein and dihydropyridine receptor purified in the absence of triadin are both retained. In contrast, triadin solubilized as a dete rgent extract of reduced triadic vesicles is retained by the hydroxyla patite column and elutes concomitantly with the junctional foot protei n and dihydropyridine receptor. These findings contrast with the obser vation that native non-reduced triadin is tightly bound to hydroxylapa tite and can be separated from the dihydropyridine receptor and the ju nctional foot protein with elevated potassium phosphate concentrations . Triadin derived from a detergent extract of reduced vesicles is reta ined with the hydroxytapatite column in the presence of 180 mM potassi um phosphate (0 KCl) which eluted a portion of the junctional foot pro tein and dihydropyridine receptor. Triadin can then be eluted with the remaining portion of junctional foot protein and dihydropyridine rece ptor upon the addition of KCl (820 mM) to the 180 mM potassium phospha te medium. Gel electrophoresis confirmed the enrichment of junctional proteins in the 180 mM KPi/820 mM KCl eluate. Rate zonal centrifugatio n of the 180 mM KPi/820 mM KCI eluate shows that a portion of triadin co-migrates with the dihydropyridine receptor indicative of a much hig her molecular weight entity than monomeric triadin. Triadin and the di hydropyridine receptor were, however, separated from the junctional fo ot protein on rate zonal centrifugation. The dissociated proteins of t he complex elute from hydroxylapatite columns similar to the purified proteins. Triadin in the high salt hydroxylapatite extract could also be immunoprecipitated by a monoclonal antibody to the junctional foot protein. Furthermore, the dihydropyridine receptor is immunoprecipitat ed by a monoclonal antibody directed against triadin providing another indication of a complex between the three proteins. Collectively, the se results demonstrate a role for triadin as the linkage between the j unctional foot protein and dihydropyridine receptor creating a ternary complex at the triad junction in skeletal muscle.