Figure 2.

Increasing Ca²⁺ influx counteracted the effects of EGTA and increased the amount of fast endocytosis. (A) Scaled capacitance responses elicited by brief stimuli in terminals loaded with 2 mM EGTA. The response in black (A_fast = 16%, τ _fast = 0.6 s, and τ_slow = 20 s) was elicited by a Ca²⁺ current of 140 pA (Inset). Larger Ca²⁺ currents (red traces) were associated with a larger proportion of fast endocytosis (85% and 63% in response to currents of 450 and 560 pA, respectively). (B) The time to half-recovery of the capacitance response (t_1/2) as a function of the Ca²⁺ current. In perforated patch recordings the kinetics of endocytosis were independent of the Ca²⁺ current (●, each n = 3). The average value of t_1/2 was 0.9 ± 0.1 s (n = 19). In terminals dialyzed with EGTA, t_1/2 was shorter for larger Ca²⁺ currents (○, each point n = 5). The line fitted to these measurements extrapolates to a t_1/2 of 6.2 s for the weakest Ca²⁺ currents. Results from terminals dialyzed with between 2 and 75 mM EGTA have been collected together because the kinetics of endocytosis were similar at all of these concentrations (see Fig. 4). Terminals treated with EGTA-AM displayed behavior similar to that seen in terminals loaded with 2 mM EGTA. (C) The percentage of membrane retrieved by fast endocytosis increased with larger Ca²⁺ currents. Single or double exponential functions were fitted to the recovery phase of the capacitance responses obtained in a total of 25 terminals loaded with 2–75 mM EGTA. In 11 terminals recovery was best described by a double exponential function, where τ_fast averaged 1.2 ± 0.2 s. In 14 terminals the whole recovery phase was adequately described with a single exponential function. In nine of these τ was longer than 3 s, and so recovery was considered all slow. In the remaining five, τ < 2 s, recovery was considered all fast.