Saturation of calcium channels and surface charge effects in skeletal muscle fibres of the frog

Abstract

Voltage-clamp and current-clamp experiments were performed to study Ca2+ and Ba2+ permeation through Ca channels in intact twitch skeletal muscle fibres of the frog. Surface charge effects were taken into consideration. Ca2+ (ICa) or Ba2+ (IBa) currents, or Ca2+ and Ba2+ action potentials were recorded in the presence of external tetraethylammonium (TEA+) ions and by replacing C1- for CH3SO3-. To further block K+ outward currents, muscles were incubated in a K+-free, TEA+ and Cs+-containing solution prior to experiments. When 10 mM-Ca2+ was replaced by 10 mM-Ba2+, the I/V curve for the peak inward current shifted by 15-20 mV to more negative potentials and the maximal peak inward current increased from -39 +/- 2 mA cm-3 (5) to -51 +/- 3 mA cm-3 (7). The decay of ICa and IBa followed a simple exponential time course and became faster for large depolarizations. The overshoot of the action potentials changed 29 +/- 3 mV or 32 +/- 3 mV for a 10-fold change in the Ca2+ or Ba2+ concentrations respectively. Ca2+ action potentials were 15-20 mV larger than Ba2+ action potentials. The maximum rate of rise Vmax and the Ca2+ or Ba2+ conductance GC2+ during the plateau tend to saturate as divalent cation concentration was increased. The Michaelis constant (Km) values obtained were respectively: 5.6 and 6.0 mM for Ca2+ and 12.5 and 8.0 mM for Ba2+. When Ca2+ or Ba2+ concentrations were increased, the effective threshold of the inward current Theff and the membrane potential E* at Vmax shifted to more positive potentials along the voltage axis. These shifts were similar for Theff and E* and were more pronounced for Ca2+ than for Ba2+. Voltage shifts could be adequately quantified by the Gouy-Chapman theory with a density of surface charges near Ca channels of 0.20 e nm-2 and including a specific binding constant for Ca2+ of 45 +/- 4 m-1. The fractional increase of the Ca2+ and Ba2+ calculated concentrations at the membrane surface near the channel was smaller than the corresponding one in the bulk solution. This partially explained the reported saturation. Saturation was still present in the Vmax of GC2+ curves corrected for surface concentration. The corrected Km values for the Vmax data were 60 mM for Ca2+ and 350 mM for Ba2+.(ABSTRACT TRUNCATED AT 400 WORDS)