Extraction of junctional complexes from triad junctions of rabbit skeletal muscle

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Anthony H. Caswell, Committee Chair


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.


Biology, General; Biophysics, General

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