Extraction of junctional complexes from triad junctions of rabbit skeletal muscle.
Triadin in skeletal muscle exists as a disulfide linked oligomer. It does not dissolve well in CHAPS detergent even in 1 M KCl, but is solubilized after reduction to its monomer by the addition of 2-mercaptoethanol. Purified reduced triadin is not retained on a hydroxylapatite column in the presence of 30 mM Potassium phosphate, while the junctional foot protein and dihydropyridine receptor purified in the absence of triadin are both retained. In contrast, triadin solubilized as a detergent extract of reduced triadic vesicles is retained by the hydroxylapatite column and elutes concomitantly with the junctional foot protein and dihydropyridine receptor. These findings contrast with the observation that native non-reduced triadin is tightly bound to hydroxylapatite and can be separated from the dihydropyridine receptor and the junctional foot protein with elevated potassium phosphate concentrations. Triadin derived from a detergent extract of reduced vesicles is retained with the hydroxytapatite column in the presence of 180 mM potassium phosphate (0 KCl) which eluted a portion of the junctional foot protein and dihydropyridine receptor. Triadin can then be eluted with the remaining portion of junctional foot protein and dihydropyridine receptor upon the addition of KCl (820 mM) to the 180 mM potassium phosphate medium. Gel electrophoresis confirmed the enrichment of junctional proteins in the 180 mM KPi/820 mM KCl eluate. Rate zonal centrifugation of the 180 mM KPi/820 mM KCl eluate shows that a portion of triadin co-migrates with the dihydropyridine receptor indicative of a much higher molecular weight entity than monomeric triadin. Triadin and the dihydropyridine receptor were, however, separated from the junctional foot protein on rate zonal centrifugation. The dissociated proteins of the 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 immunoprecipitated by a monoclonal antibody directed against triadin providing another indication of a complex between the three proteins. Collectively, these results demonstrate a role for triadin as the linkage between the junctional foot protein and dihydropyridine receptor creating a ternary complex at the triad junction in skeletal muscle.
Motoike
H K
HK
Department of Molecular & Cellular Pharmacology, University of Miami School of Medicine, FL 33101.
Caswell
A H
AH
Smilowitz
H M
HM
Brandt
N R
NR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D015220:Calcium Channels / Q000737:chemistry / Q000302:isolation & purification*; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D002501:Centrifugation, Zonal; D002793:Cholic Acids; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D012995:Solubility
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D002793:Cholic Acids; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C498270:Trdn protein, mouse; C070048:triadin; D017886:Durapatite; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate