Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the 1,4-dihydropyridine photoaffinity probe, [3H]azidopine.
The arylazide 1,4-dihydropyridine, [3H]azidopine, binds with high affinity to calcium channels in partially purified guinea-pig skeletal muscle transverse tubule membranes. Upon brief exposure to u.v. light, [3H]azidopine incorporates covalently into transverse tubule membrane proteins, as judged by SDS-PAGE. After alkylation of sulfhydryl groups with N-ethylmaleimide three specifically labelled bands of mol wts. 240 kd, 158 kd and 99 kd are always observed with fluorography after one-dimensional SDS-PAGE. Two other specific bands with mol. wts. of 52 kd and 55 kd, respectively, were sometimes observed. Two-dimensional SDS-PAGE (non-reduced but alkylated in the first dimension and reduced in the second dimension) revealed that the 240-kd band after reduction migrates with a mol. wt. of 99 kd. The 158-kd and 99-kd bands do not change in mobility. It is suggested that [3H]azidopine binds in such a way that the arylazide moiety of the ligand comes into contact with at least three calcium channel components: the A component of mol. wt. 240 kd, the B component of mol. wt. 158 kd and a C component of mol. wt. 99 kd. B and C are non-covalently bonded subunits of the channel, whereas A could be a heterodimer consisting of B and C, linked by disulfide bonds. Subunits of smaller mol. wt. may be also part of the ionic pore. Photolabelling of transverse tubule membranes after high energy irradiation with 10 MeV electrons supports this interpretation.
D R
DR
Ferry
K
K
Kämpf
A
A
Goll
H
H
Glossmann
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure; D004095:Dihydropyridines; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D007473:Ion Channels / Q000032:analysis* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis*; D008968:Molecular Conformation; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D011725:Pyridines; D014316:Tritium
D000345:Affinity Labels; D001386:Azides; D004095:Dihydropyridines; D007473:Ion Channels; D008565:Membrane Proteins; D011725:Pyridines; D014316:Tritium; C041130:azidopine