Competition between sodium and calcium ions in transmitter release at mammalian neuromuscular junctions

1. Frequencies of miniature end-plate potentials (m.e.p.p.s) were recorded at neuromuscular junctions in rat diaphragm-phrenic nerve preparations in vitro.2. In the presence of raised [K] (15-20 mM) lowering [Na] caused a rapid increase in m.e.p.p. frequency whether [Ca] was low or normal. Raising [Na] towards the normal concentration (162 mM) caused a slow fall in frequency and raising [Ca] in the range 0.32-2 mM caused a slow increase in frequency. These effects were less in the normal [K] (5 mM).3. Mean m.e.p.p. frequencies were determined for solutions containing 15 mM-K and combinations of [Ca] and [Na]. M.e.p.p. frequency varied inversely with [Na] when [Ca] was constant. In each of the three Na concentrations used (162, 113 and 65 mM) raising [Ca] in the range 0.32-2 mM increased m.e.p.p. frequency but when raised above 2-3 mM, Ca depressed frequency.4. A model was proposed in which Ca affected transmitter release by changing the concentration in the presynaptic membrane of a complex CaX to which the rate of transmitter release was directly proportional. Higher concentrations of Ca depressed transmitter release by inactivating CaX. Sodium ions competitively depressed release either by competing with calcium ions for association with X or by reducing the affinity of X for Ca.5. When [Na] was lowered in solutions containing raised [Mg] and [Ca], the increase of mean m.e.p.p. frequency was greater than that observed in raised [Ca] and normal [Mg] and was of the same order as the increases seen in low [Ca]. The result was interpreted to indicate either that Na and Mg do not compete with Ca at the same site or that Mg affects the affinity of X for Ca and Na.6. The effect of lowering [Na] on m.e.p.p. frequency was a specific effect of Na ions. When LiCl was substituted for NaCl, the increase of m.e.p.p. frequency persisted. Changes in [Cl] had no effect on m.e.p.p. frequency.7. There was a linear relation between the mean logarithm of m.e.p.p. frequencies and [K], the slope of the relation increasing as [Na] was lowered. Conversely, lowering [Na] caused a greater increase in m.e.p.p. frequency as [K] was raised.8. The variation of m.e.p.p. frequencies in a diaphragm was roughly proportional to a second or higher power of [Na] and inversely proportion to [Ca]. It was thought that this could be due to differences in chelation of Ca which were more apparent at low Ca concentrations.9. The similarities between the effects of Na, Ca and K on m.e.p.p. frequency and the effects of these ions on Ca-influx in heart muscle led to the suggestion that transmitter release is proportional to the concentration of a negatively charged complex of a carrier X with one calcium ion (CaX) at the internal surface of the membrane and that changes in membrane potential affect transmitter release by changing the distribution or location of CaX in the membrane.