(A) Presynaptic action potentials and evoked IPSCs recorded immediately after presynaptic BC break-in (left), 20 min after break-in to load 3 mM BAPTA into presynaptic terminals (middle), and after the presynaptic recoding pipette was changed to one containing control solution (right). 10 consecutive individual traces (gray) and the corresponding average trace (red) are shown superimposed. (B) Time course of the effect of 1 mM (○, 5 pairs), 3 mM (△, 4 pairs) and 10 mM (▿, 5 pairs) BAPTA. Data from each pair were normalized to the average amplitude obtained from the first 20 consecutive traces and averaged across pairs. (C) Similar recording as shown in (A), except that the internal solution for the presynaptic BC contained 30 mM EGTA. (D) Time course of the effect of 0.1 mM (○, control, 4 pairs), 10 mM (△, 5 pairs) and 30 mM (▿, 6 pairs) EGTA. In all experiments included in (B) and (D), the presynaptic BC was patched twice to demonstrate recovery. Symbols indicate mean, error bars represent SEM. (E) Plot of steady-state effects of chelators on IPSC peak amplitude against chelator concentration for BAPTA (red circles) and EGTA (blue circles). Curves represent Hill equations fit to the data points. IC50 values were 0.6 mM and 16.0 mM, Hill coefficients were 0.96 and 1.51, respectively. (F) Same data, fit with linearized models of Ca2+ diffusion and buffering (continuous curve, constant coupling distance; small dashing, half-normally distributed coupling distance; large dashing, skewed-normally distributed coupling distance; corresponding distributions shown as insets on the right). For the single-channel model, the coupling distance was estimated as 11.4 nm. For the half-normal distribution, the standard deviation was 10.5 nm (expectation value 13.9 nm; skewness 1.0). For the skewed-normal distribution, location, scale, and shape parameter were 13.7 nm, 8.6 nm, and −1.11, respectively (expectation value 10.1 nm; skewness 0.40). (G) Statistical errors. Histogram of coupling distance estimates in 1000 bootstrap replications. (H) Systematic errors. Plot of coupling distance against resting Ca2+ concentration and endogenous buffer product. Red point indicates default parameter values. In (G) and (H), the model with constant coupling distance was used. All experiments were performed at ∼22°C.