AbstractThe impact of presynaptic transmitter release site organization on synaptic function has been a vibrant area of research for synaptic physiologists. Because there is a highly nonlinear relationship between presynaptic calcium influx and subsequent neurotransmitter release at synapses, the organization and density of calcium sources (voltage‐gated calcium channels [VGCCs]) relative to calcium sensors located on synaptic vesicles is predicted to play a major role in shaping the dynamics of neurotransmitter release at a synapse. Here we review the history of structure‐function studies within transmitter release sites at the neuromuscular junction across three model preparations in an effort to discern the relationship between VGCC organization and synaptic function, and whether that organizational structure imparts evolutionary advantages for each species.