Single-Pixel Optical Fluctuation Analysis of Calcium Channel Function in Active Zones of Motor Nerve Terminals

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca2+) channels during an action potential and the number of such Ca2+ channels within active zones of frog neuromuscular junctions. Analysis revealed similar to 36 Ca2+ channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (similar to 200-250). The probability that each channel opened during an action potential was only similar to 0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca2+ channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca2+ influx through individual Ca2+ channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones.