Dihydropyridine receptors in transverse tubules from normal and dystrophic chicken skeletal muscle.
Calcium overload is a fundamental pathogenic event associated with chronic muscle degeneration in muscular dystrophies. The possibility that L-type voltage-dependent calcium channels were involved in the etiology of chicken muscular dystrophy was investigated by studying the dihydropyridine receptors in transverse tubule membranes isolated from skeletal muscle of normal (line 412) and dystrophic (line 413) chickens. The yield of T-tubular protein from dystrophic muscle was considerably increased compared with that from normal muscle (2.51 +/- 0.18 vs 1.04 +/- 0.31 mg protein x 100 g muscle-1). The binding of the calcium channel antagonist (+) [3H]PN200-110 to the dihydropyridine receptor in transverse tubule preparations was relatively slow, markedly affected by temperature and required divalent cations. (+) [3H]PN200-110 equilibrium binding assays revealed a single class of high-affinity sites and showed that maximum binding capacity (Bmax) (3.17 +/- 0.47 for normal and 3.51 +/- 0.52 pmol x mg protein-1 for dystrophic transverse tubules) and dissociation constant (Kd) (0.32 +/- 0.07 and 0.26 +/- 0.09 nM, respectively) were not significantly different in normal and dystrophic membranes. Kinetic studies indicated that normal and dystrophic transverse tubules did not differ significantly in association (2.54 x 10(6) and 2.27 x 10(6) M(-1)s(-1), respectively) and dissociation (8.5 x 10(-4) and 9.3 x 10(-4)s(-1), respectively) rate constants. Since dissociation kinetics for both preparations were monoexponential under all the experimental conditions employed, no low-affinity binding sites for (+) [3H]PN200-110 could be detected in chicken transverse tubules membranes. However, immunoblot assay, using a monoclonal antibody, revealed that dystrophic transverse tubules as compared with normal membranes were enriched twofold with the alpha 1-subunit of the dihydropyridine receptor. Therefore, although dihydropyridine-binding sites were not altered in transverse tubule membranes from dystrophic chicken skeletal muscle, both the increased yield in T-tubule vesicles and the enhanced immunodetection of the alpha 1-subunit of the dihydropyridine receptor, suggest that total content in dihydropyridine receptor is higher in dystrophic than in normal muscle.
Moro
G
G
Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Complutense University, Madrid, Spain.
Saborido
A
A
Delgado
J
J
Molano
F
F
Megias
A
A
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology* / Q000648:ultrastructure; D020746:Calcium Channels, L-Type; D002462:Cell Membrane / Q000648:ultrastructure; D002645:Chickens; D017275:Isradipine / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000648:ultrastructure*; D009124:Muscle Proteins / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology; D009137:Muscular Dystrophy, Animal / Q000473:pathology; D011485:Protein Binding / Q000502:physiology; D013696:Temperature; D014316:Tritium
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D014316:Tritium; D008274:Magnesium; D002118:Calcium; D017275:Isradipine