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 dihyd ropyridine receptor (DKPR) 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 whe ther the lack of the DHPR in dysgenic skeletal muscle results in a fai lure of triad formation, using eel lines (GLT and NLT) derived from dy sgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Larg e T antigen, Both cell lines express muscle-specific proteins and begi n organization of sarcomeres as demonstrated by immunocytochemistry. S imilar to primary cultures, dysgenic (GLT) myoblasts show a higher inc idence of cell fusion than their normal counterparts (NLT). NLT myotub es develop spontaneous contractile activity, and fluorescent Ca2+ reco rdings show Ca2+ release in response to depolarization. In contrast, G LTs show neither spontaneous nor depolarization-induced Ca2+ transient s, but do release Ca2+ from the sarcoplasmic reticulum (SR) in respons e to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha(1) subunit of the skeletal muscle DHPR, an d the alpha(2) subunit is mistargeted. Nevertheless, the ryanodine rec eptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR a subunits in the T-tubules, In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced fe et, These findings provide conclusive evidence that interactions betwe en the DHPR and RyR are not involved in the formation of triad junctio ns or in the normal organization of the RyR in the junctional SR.