Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle.
The isolated dihydropyridine receptor and junctional foot protein were employed as protein ligands in overlay experiments to investigate the mode of interaction of these two proteins. As previously demonstrated by Brandt et al. [Brandt et al. (1990) J. Membr. Biol. 113, 237-251], the DHP receptor directly binds to an intrinsic terminal cisterna protein of Mr 95,000 (95-kDa protein). The junctional foot protein also binds to an Mr 95,000 protein showing similar organelle distribution to the 95-kDa protein which binds to the dihydropyridine receptor. The 95-kDa protein which binds to the dihydropyridine receptor was isolated to over 85% purity employing sequential column chromatography. Junctional foot protein and dihydropyridine receptor overlays of the column fractions at successive stages of isolation show an identical pattern of distribution, indicating that both probes bind to the same protein. When CHAPS-solubilized terminal cisterna/triads were passed through Sepharose with attached 95-kDa protein, the junctional foot protein was specifically retained, as evidenced by ryanodine binding. The junctional foot protein was incompletely released by 1 M NaCl. The alpha 1 subunit but not the beta subunit of the dihydropyridine receptor was also specifically retained, as evidenced by immunoblotting employing dihydropyridine receptor subunit-specific antibodies. A 170-kDa Stains-all blue staining protein, which appears to be bound to the luminal side of the terminal cisterna, was also retained on the 95-kDa protein column. From these findings, a model for the triad junction is proposed.
Kim
K C
KC
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
Caswell
A H
AH
Talvenheimo
J A
JA
Brandt
N R
NR
D000818:Animals; D002121:Calcium Channel Blockers / Q000737:chemistry / Q000378:metabolism*; D015220:Calcium Channels; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003300:Copper; D008958:Models, Molecular; D009132:Muscles / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D019837:Ryanodine Receptor Calcium Release Channel; D003300:Copper