Voltage-Activated Calcium Signals in Myotubes Loaded with High Concentrations of EGTA

Abstract

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.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1

“…This protocol provides six long relaxation intervals after the single pulse and following the tetani that can be used to characterize Ca 2ϩ removal (19,20,42). After data reduction by averaging every five consecutive sample points, traces were smoothed using an adaptive digital filter (35). A Ca 2ϩ removal model including binding and transport was used to simultaneously fit the relaxation phases in the long intervals and in the 700-ms recording period following the last tetanus.…”

IQ-Motif Proteins Influence Intracellular Free Ca2+in Hippocampal Neurons Through Their Interactions With Calmodulin

“…For this reason, we chose to plot the "fitted curve" as circle/crosses (at discrete time points) and overlay them with the simulation results (solid/dotted lines). Ca 2ϩ -binding kinetics of EGTA used in the simulation were as follows: EGTA binds Ca 2ϩ with a 1:1 stoichiometry and the Ca 2ϩ association and dissociation rates are 55.8 M Ϫ1 ⅐ s Ϫ1 and 2.12 s Ϫ1 , respectively (Schuhmeier et al 2003). …”

Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes

“…The computer routine determined the maximal transport rate of the pump (PV max ) from the declining phase of the calcium transient following the stimulation. All other parameters were held constant at values taken from the literature [26]. A single fit was carried out for each myotube and then used for all calcium transients on the given cell.…”