Ca2+ inward current and fura‐2 Ca2+ transients were simultaneously recorded in porcine myotubes. Myotubes from normal pigs and cells from specimens homozygous for the Arg615Cys (malignant hyperthermia) mutation of the skeletal muscle ryanodine receptor RyR1 were investigated. We addressed the question whether this mutation alters the voltage dependence of Ca2+ release from the sarcoplasmic reticulum. The time course of the total flux of Ca2+ into the myoplasm was estimated. Analysis showed that the largest input Ca2+ flux occurred immediately after depolarization. Amplitude and time course of the Ca2+ flux at large depolarizations were not significantly different in the Arg615Cys myotubes. Ca2+ release from the sarcoplasmic reticulum was activated at more negative potentials than the L‐type Ca2+ conductance. In the controls, the potentials for half‐maximal activation (V½) were ‐9.0 mV and 16.5 mV, respectively. In myotubes expressing the Arg615Cys mutation, Ca2+ release was activated at significantly lower depolarizing potentials (V½= ‐23.5 mV) than in control myotubes. In contrast, V½ of conductance activation (13.5 mV) was not significantly different from controls. The specific shift in the voltage dependence of Ca2+ release caused by this mutation can be well described by altering a voltage‐independent reaction of the ryanodine receptor that is coupled to the voltage‐dependent transitions of the L‐type Ca2+ channel.

The transverse tubules of skeletal muscle cells contain a high density of DHP binding sites (reviewed by Lamb, 1992). Two functions have been assigned to the DHPbinding proteins (DHP receptors): the activation of a Ca 2+ conductance (L-type Ca 2+ current) with exceptionally slow kinetics, and the activation of intracellular release of Ca 2+ from the sarcoplasmic reticulum (SR) (reviewed in Melzer et al. 1995). The latter process is probably the result of direct interaction between DHP receptor and Ca 2+ -release channels (ryanodine receptors) (reviewed in Melzer & Dietze, 2001) and does not depend on Ca 2+ entering the cell during the depolarization (Spiecker et al. 1979; Brum et al. 1988; Dirksen & Beam, 1999 & De Waard, 1998). a 1S and b have been shown to be essential for E-C coupling (Beam et al. 1986;Gregg et al. 1996) and their elimination is lethal. The fact that the y subunit (y1 isoform) could only be detected in skeletal muscle also suggests a role for this polypeptide that is important for muscle function (Jay et al. 1990;Powers et al. 1993;Wissenbach et al. 1998). y1 is a transmembrane protein of 32 kDa (222 amino acid residues) containing four putative membrane-spanning segments (Bosse et al. 1990; Jay et al. 1990). Functional studies of the y1 subunit have up to now been restricted to measuring transmembrane ionic currents. The effects of y1 on Ca 2+ or Ba 2+ inward currents have been investigated by coexpression of the subunit with the cardiac muscle a 1C subunit in heterologous expression systems (Singer et al. 1991;Wei et al. 1991;Lerche et al. 1996; Eberst et al. 1997;Sipos et al. 2000). An alternative approach to studying the role of subunits is the use of specific knockout systems. A recently generated y1-deficient Excitation-contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit y1 1. In skeletal muscle, dihydropyridine (DHP) receptors control both Ca 2+ entry (L-type current) and internal Ca 2+ release in a voltage-dependent manner. Here we investigated the question of whether elimination of the skeletal muscle-specific DHP receptor subunit y1 affects excitation-contraction (E-C) coupling. We studied intracellular Ca 2+ release and force production in muscle preparations of a mouse deficient in the y1 subunit (y_/_). The rate of internal Ca2+ release at large depolarization (+20 mV) was determined in voltageclamped primary-cultured myotubes derived from satellite cells of adult mice by analysing fura-2 fluorescence signals and estimating the concentration of free and bound Ca 2+. On average, y_/_ cells showed an increase in release of about one-third of the control value and no alterations in the time course.3. Voltage of half-maximal activation (V 1/2 ) and voltage sensitivity (k) were not significantly different in y_/_ myotubes, either for internal Ca 2+ release activation or for the simultaneously measured L-type Ca 2+ conductance. The same was true for maximal Ca 2+ inward current and conductance.4. Contractions evoked by electrical stimul...

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.

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. 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. 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. 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. 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.