Calcium dependence of evoked transmitter release at very low quantal contents at the frog neuromuscular junction.

The relationship between the rate of evoked transmitter release and the extracellular concentration of Ca ions, [Ca²⁺]₀, was studied at surface neuromuscular junctions of the frog cutaneous pectoris muscle. The average quantal content of the end-plate potential was reduced to low levels by reducing [Ca²⁺]₀ and adding 2 mM-Mn²⁺, 4 mM-Co²⁺, or 10 mM-Mg²⁺. When the motor nerve was stimulated at a low frequency (0.5-2 Hz) in 2 mM-Mn²⁺ or 4 mM-Co²⁺, the average quantal content of evoked release was proportional to the fourth power of [Ca²⁺]₀ down to the lowest measurable quantal contents, around 2-4 quanta per 1000 stimuli. Combined with previous studies, this result indicates that evoked transmitter release has a steep, nonlinear dependence on [Ca²⁺]₀ over four orders of magnitude of evoked release. Calculations predict that if evoked and spontaneous release have the same fourth power dependence on intracellular [Ca₂₊], then the curve relating evoked release and [Ca²⁺]₀ should become much less steep as the evoked release rate approaches the spontaneous release rate. Our observation that the relationship between evoked release and [Ca²⁺]₀ remains fourth power down to very low release rates suggests that most spontaneous quantal release does not have the same dependence on intracellular [Ca²⁺], or does not use the same intracellular Ca²⁺ pool, as evoked release. In 2-10 mM-Mg²⁺, the lowest average quantal contents were markedly higher than the fourth power prediction. This discrepancy may occur either because Mg²⁺ somehow elevates intracellular [Ca²⁺], or because Mg²⁺ is itself a weak activator of transmitter release. Even at very low rates of evoked release, increasing the stimulus frequency to 5-50 Hz caused a progressive increase in both evoked release and the rate of 'background' quantal release during the interstimulus interval. The frequency-dependent enhancement of both evoked and background release was more pronounced in solutions containing 10 mM-Mg²⁺ than in solutions containing 2 mM-Mn²⁺.