Ca2+ from one or two channels controls fusion of a single vesicle at the frog neuromuscular junction

Neurotransmitter release is triggered by the cooperative action of approximately five Ca2+ ions entering the presynaptic terminal through Ca2+ channels. Depending on the organization of the active zone (AZ), influx through one or many channels may be needed to cause fusion of a vesicle. Using a combination of experiments and modeling, we examined the number of channels that contribute Ca2+ for fusion of a single vesicle in a frog neuromuscular AZ. Wecompared Ca2+ influx to neurotransmitter release by measuring presynaptic action potential- evoked (AP- evoked) Ca2+ transients simultaneously with postsynaptic potentials. Ca2+ influx was manipulated by changing extracellular [Ca2+] ( Caext) to alter the flux per channel or by reducing the number of open Ca2+ channels with omega-conotoxin GVIA (omega-CTX). When Caext was reduced, the exponent of the power relationship relating release to Ca2+ influx was 4.16 +/- 0.62 ( SD; n = 4), consistent with a biochemical cooperativity of similar to 5. In contrast, reducing influx with omega- CTX yielded a power relationship of 1.7 +/- 0.44 ( n = 5) for Caext of 1.8mM and 2.12 +/- 0.44 for Caext of 0.45mM ( n = 5). Using geometrically realistic Monte Carlo simulations, we tracked Ca2+ ions as they entered through each channel and diffused in the terminal. Experimental and modeling data were consistent with two to six channel openings per AZ per AP; the Ca2+ that causes fusion of a single vesicle originates from one or two channels. Channel cooperativity depends mainly on the physical relationship between channels and vesicles and is insensitive to changes in the nongeometrical parameters of our model.