The corresponding rate constant determined between 12 and 24 degrees C strongly depended on temperature, as expected for a gating process. The decay phase of ICa under a maintained depolarization in incubated muscles was fitted by a single exponential. Tubular space clamp was improved by recording ICa from small fibres with 20-30 microns radius. 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. 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. The amplitude and time course of slow, outward currents were not obviously modified by replacing Ca2+ with Mg2+, having the two described phases. The inward current was greatly reduced or abolished by the adding of 2 mM-Cd2+ or by replacing external Ca2+ with Mg2+. 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. 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. Contraction was blocked by recording in hypertonic sucrose solutions. 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.
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