Voltage-activated calcium signals in myotubes loaded with high concentrations of EGTA.
In the present study we describe the analysis of optically recorded whole cell Ca(2+) transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca(2+) mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca(2+)-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca(2+) to the myoplasmic space (Ca(2+) input flux). The validity of the procedure was confirmed by model simulations using artificial Ca(2+) input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca(2+) input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca(2+) flux carried by the L-type Ca(2+) channels, indicating that it consists mainly of voltage-controlled Ca(2+) release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca(2+) inward current, indicating that Ca(2+) release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca(2+) release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.
Schuhmeier
R P
RP
Universität Ulm, Abteilung für Angewandte Physiologie, D-89069 Ulm, Germany.
Dietze
B
B
Ursu
D
D
Lehmann-Horn
F
F
Melzer
W
W
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003198:Computer Simulation; D004533:Egtazic Acid / Q000378:metabolism* / Q000494:pharmacology; D005470:Fluorometry / Q000379:methods; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology*
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004533:Egtazic Acid; D002118:Calcium