Transmitter release is evoked with low probability predominately by calcium flux 2 through single channel openings at the frog neuromuscular junction

39 The quantitative relationship between presynaptic calcium influx and transmitter 40 release critically depends on the spatial coupling of presynaptic calcium channels to 41 synaptic vesicles. When there is a close association between calcium channels and 42 synaptic vesicles, the flux through a single open calcium channel may be sufficient to 43 trigger transmitter release. With increasing spatial distance, however, a larger number 44 of open calcium channels might be required to contribute sufficient calcium ions to 45 trigger vesicle fusion. Here we used a combination of pharmacological calcium channel 46 block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo 47 reaction-diffusion simulations in the adult frog neuromuscular junction, to show that 48 release of individual synaptic vesicles is predominately triggered by calcium ions 49 entering the nerve terminal through the nearest open calcium channel. Furthermore, 50 calcium ion flux through this channel has a low probability of triggering synaptic vesicle 51 fusion (~6%), even when multiple channels open in a single active zone. These 52 mechanisms work to control the rare triggering of vesicle fusion in the frog 53 neuromuscular junction from each of the tens of thousands of individual release sites at 54 this large model synapse. 55