Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle.
Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.
Powell
J A
JA
Department of Biological Sciences, Smith College, Northampton, Massachusetts 01063, USA.
Petherbridge
L
L
Flucher
B E
BE
D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D002460:Cell Line; D008561:Membrane Fusion; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel