Voltage-controlled Ca2+ release in normal and ryanodine receptor type 3 (RyR3)-deficient mouse myotubes.
1. Primary cultured myotubes were derived from satellite cells of the diaphragm obtained from both normal mice (RyR3+/+) and mice with a targeted mutation eliminating expression of the type 3 isoform of the ryanodine receptor (RyR3-/-). Using the whole-cell patch clamp technique, L-type Ca2+ currents were measured during step depolarizations. Simultaneously, intracellular Ca2+ transients were recorded with the fluorescent indicator dye fura-2. 2. After correction for non-instantaneous binding of Ca2+ to the indicator dye and taking into account the dynamics of Ca2+ binding to intracellular constituents, an estimate of the time course of the Ca2+ release rate from the sarcoplasmic reticulum (SR) was obtained. 3. The calculated SR Ca2+ release flux exhibited a marked peak within less than 12 ms after the onset of the voltage-clamp depolarization and fell rapidly thereafter to a five times lower, almost steady level. It declined rapidly after termination of the depolarization. 4. Signals in normal and RyR3-deficient myotubes showed no significant difference in the activation of Ca2+ conductance and in amplitude, time course and voltage dependence of the Ca2+ efflux from the SR. 5. In conclusion, the characteristics of voltage-controlled Ca2+ release reported here are similar to those of mature mammalian muscle fibres. In contrast to differences observed in the contractile properties of RyR3-deficient muscle fibres, a contribution of RyR3 to excitation-contraction coupling could not be detected in myotubes.
Department of Applied Physiology, University of Ulm, D-89069 Ulm, Germany.
B
B
Dietze
F
F
Bertocchini
V
V
Barone
A
A
Struk
V
V
Sorrentino
W
W
Melzer
D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D003964:Diaphragm / Q000166:cytology / Q000502:physiology; D004558:Electric Stimulation; D004594:Electrophysiology; D005456:Fluorescent Dyes; D016257:Fura-2; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D019837:Ryanodine Receptor Calcium Release Channel / Q000172:deficiency* / Q000235:genetics
D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium; D016257:Fura-2