Transmitter induced calcium entry across the post-synaptic membrane at frog end-plates measured using arsenazo III.

Abstract

1. The Ca2+ influx occurring across the post-synaptic membrane during transmitter action was studied at the frog neuromuscular junction, using the Ca sensitive dye arsenazo III to monitor the resulting changes in free myoplasmic Ca2+ concentration. 2. Calibration experiments showed a linear relationship between the amount of Ca2+ injected by ionophoresis into a muscle fibre, and the peak size of the arsenazo light absorbance record. 3. Ionophoretic application of acetylcholine (ACh) to voltage clamped end-plates gave rise to an arsenazo signal. The size of this response varied with the Ca2+ concentration in the bathing solution. 4. The arsenazo light response increased in size steeply, and non-linearly, with hyperpolarization of the end-plate membrane, even when the end-plate current increased approximately linearly with hyperpolarization. The voltage dependence of the light response could be fitted well by an exponential with a voltage constant of 28 mV. Changes in Ca2+ concentration of the bathing medium had little effect on this relationship. 5. At end-plates bathed in isotonic CaCl2 solution the voltage dependence of both the arsenazo light response, and the end-plate current showed a closely similar, non-linear relationship. 6. Addition of 12 mM-Co2+ to a bathing solution initially containing 12 mM-Ca2+ substantially reduced the size of the arsenazo light response, and the voltage dependence of this response became more linear. 7. Arsenazo light responses were also recorded in response to transmitter release evoked by nerve stimulation. The size of the nerve evoked light response showed a non-linear voltage dependence, whilst the end-plate current was a linear function of membrane potential.