Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.

Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca2+ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cd2+e or with an "intracellular", EGTA-buffered solution containing less than 0.1 microM Ca2+e. Pulse application and recording were by a perfused macro-patch-clamp electrode. The muscle outside the electrode (bath) was superfused with Ringer's solutions containing Cd2+b to block Ca2+ inflow and normal (1.8 mM) or elevated (10 mM) Ca2+b. The depolarization level of the terminal during current pulses that generated maximal Ca2+ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca2+ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca2+ inflow is high. However, for large depolarizations (> 1.5 relative units), the depression of release by block of Ca2+ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca2+ inflow. If the extra-electrode superfusion of Ca2+b of the muscle was elevated to 10 mM and Cd2+b was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1 microM Ca2+e raised the average release paradoxically. With 0.5 mM Cd2+b this paradoxical increase of release was, on average, 4-fold at 6 degrees C, and 19-fold at 16 degrees C. Quantal endplate currents recorded in less than 0.1 microM Ca2+e had slightly increased amplitudes, and decay time constants were prolonged by about 50%. The results are interpreted to support the Ca2+/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca2+ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca2+ concentration is sufficiently high, large depolarizations can elicit release independent of the presence or absence of Ca2+ inflow.