Separation of charge movement components in mammalian skeletal muscle fibres

1. Intramembrane charge movements, I(ICM), were measured in rat skeletal muscle fibres in response to voltage steps from a -90 mV holding potential to a wide test voltage range (-85 to 30 mV), using a double Vaseline-gap voltage-clamp technique. Solutions were designed to minimise ionic currents. Ca(2+) current was blocked by adding Cd(2+) (0.8 mM) to the external solution. In a subset of experiments Cd(2+) was omitted to determine which components of the charge movement best correlated with L-type Ca(2+) channel gating. 2. Detailed kinetic analysis of I(ICM) identified two major groups of charges. The first two components, designated Q(a) and Q(b), were the only charges moved by small depolarising steps. The second group of components, Q(c) and Q(d), showed a more positive voltage threshold, -35.6 +/- 2.0 mV, (n = 6) in external solution with Cd(2+), and -41.1 +/- 2.0 mV (n = 12) in external solution without Cd(2+). Notably, in external solution without Cd(2+) the voltage threshold of Ca(2+) current, I(Ca), activation had a similar value, being -38.1 +/- 2.4 mV. 3. The sum of three Boltzmann functions, Q(1), Q(2) and Q(3), showing progressively more positive transition voltages, could be fitted to charge versus voltage, Q(ICM)-V, plots. The three Boltzmann terms identified three charge components: Q(1) described the shallow voltage-dependent Q(a) and Q(b) charges, Q(2) and Q(3) described the steep voltage-dependent Q(c) and Q(d) charges. 4. In external solution without Cd(2+) the charge kinetics changed: a slow decaying phase was replaced by a pronounced delayed hump. Moreover, the transition voltages of the individual steady-state charge components were shifted towards negative potentials (from 6.3 to 8.2 mV). Nevertheless, the overall charge and steepness factors were conserved. 5. In conclusion, these experiments allowed a clear separation of four components of intramembrane charge movements in rat skeletal muscle, showing that there are no fundamental differences with respect to charge movement components between amphibian and mammalian twitch muscle. Moreover, Q(c) and Q(d) charge are correlated with L-type Ca(2+) channel gating.