Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro.
Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95-kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential-induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.
Flucher
B E
BE
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Andrews
S B
SB
Fleischer
S
S
Marks
A R
AR
Caswell
A
A
Powell
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000302:isolation & purification / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D051379:Mice; D008817:Mice, Mutant Strains; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C498270:Trdn protein, mouse; C498271:Trdn protein, rat; C070048:triadin; D000252:Calcium-Transporting ATPases; D002118:Calcium