(A) Diagram of the site activation mechanism. Three states are introduced: [I], inactive, [D], delay, and [A], activated. SV fusion is only allowed from sites in state [A]. The rate from [I] to [D] is increased by Ca2+, whereas the rate from [D] to [A] is slower and independent of Ca2+, thereby introducing a delay. All (in)activation reactions are assumed to be reversible. (B) Full site activation model. The Ca2+ (un)binding of the SVs follow the same equations as in the single-sensor model and occurs independently of the site (in)activation. Replenishment is allowed into empty release sites regardless of activation status. (C) Average, experimental eEJC traces (black) together with average simulated traces (red). (D) eEJC1 amplitudes of experiment (black) and simulation (red). Error bars and colored bands show standard deviation. Like with the three models described in the main text, simulations reproduce eEJC1 amplitudes well. (E) Average, normalized eEJC traces of experiment (black) and simulation (red). (F) PPR values of experiment (gray) and simulation (blue). Error bars and colored bands show standard deviation. Like the unpriming model, simulations reproduce the experimentally observed facilitation. (G) Average simulated traces (red) and examples of different outcomes of the stochastic simulation (colors). (H) Plot of the mean synaptic variance vs. the mean eEJC1 values, both from the experiment (black) and the simulation (red). The curves show the best fitted parabolas with forced intercept at (0,0) (simulation: Var = −0.0043*< eEJC1>2+0.5398 nA*< eEJC1>, corresponding to nsites = 233 and q = 0.54 nA when assuming a classical binomial model (Clements and Silver, 2000), see Materials and methods). Like in experiments, simulations lead to decreasing variance at the highest Ca2+ concentrations. (I) The number of sites in state [I] and [A] (gray and red resp.) in simulations with extracellular Ca2+ concentrations of 0.75 mM and 10 mM (solid and dashed resp.). The varying basal Ca2+ concentration yield different initial amounts of site activation. The activation of sites mainly occurs between APs because of the delay state. Experimental data (example traces and means) depicted in panels C-F,H are replotted from Figure 2A–D, F. Parameter values used for simulations can be found in Tables 1–3. Simulation scripts can be found in Source code 1. Results from simulations (means and SDs) can be found in the accompanying source data file (Figure 7—source data 1).