Calcium-channel gating in frog skeletal muscle membrane: effect of temperature

Abstract

Voltage-clamp experiments using the three micro-electrode method were performed to study the temperature dependence of the calcium current ICa in intact twitch skeletal muscle fibres of the frog. Contraction was blocked by recording in hypertonic sucrose solutions. For depolarizations smaller than 0 mV the decay of the transient, slow, inward current, recorded in the presence of external tetraethylammonium (TEA+) and by replacing Cl- for CH3SO3-, followed a complex time course. For larger depolarizations, after the initial inward current, there was a prominent, slow, outward current which showed two phases: after reaching a peak (time to peak 1.0 sec, peak amplitude 20-50 microA/cm2 at 20 mV) it slowly declined to a steady level in about 2-3 sec at 23 degrees C. The inward current was greatly reduced or abolished by the adding of 2 mM-Cd2+ or by replacing external Ca2+ with Mg2+. The amplitude and time course of slow, outward currents were not obviously modified by replacing Ca2+ with Mg2+, having the two described phases. However, in the presence of Cd2+ the first transient phase of the outward current was not detected and only outward currents slowly increasing to a steady level were observed. Reliable ICa records were obtained by further blocking K+ outward currents by incubating the muscles in a K+-free TEA+- and Cs+-containing solution prior to experiments. Tubular space clamp was improved by recording ICa from small fibres with 20-30 microns radius. The decay phase of ICa under a maintained depolarization in incubated muscles was fitted by a single exponential. The corresponding rate constant determined between 12 and 24 degrees C strongly depended on temperature, as expected for a gating process. The values for the activation energy and the corresponding Q10 (calculated for a 10-20 degrees C transition) were respectively: 17.5 +/- 1.0 kcal/mole and 2.9 +/- 0.2 at 0 mV, and 18.0 +/- 1.5 kcal/mole and 3.0 +/- 0.3 at -20 mV. The activation phase of ICa, analysed following the m alpha h Hodgkin-Huxley kinetic model, showed a similar temperature dependence with a Q10 of 3.0 +/- 0.3. The peak amplitude of ICa and the limiting Ca2+ permeability had a lower Q10 value of about 1.6. For a given temperature the rate constant of decay was independent of ICa peak amplitude in disagreement with a current-dependent process (intratubular Ca2+ depletion or intracellular Ca2+ accumulation) for the decay of ICa. In conclusion, our results favour a gating process (inactivation) as the principal mechanism underlying the decay phase of ICa under a maintained depolarization.