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. 2001 Dec 4;98(26):15282–15287. doi: 10.1073/pnas.261311698

Figure 1.

Figure 1

Calcium buffers caused a proportion of synaptic vesicles to be retrieved by slow endocytosis. (A) Examples of capacitance responses elicited by a 20-ms depolarization to −10 mV (delivered at arrow), recorded by using the perforated patch technique. Compared are single responses in untreated terminals (black) and terminals incubated in EGTA-AM (red). (B) Responses from A scaled to the same maximum to allow direct comparison of the kinetics of endocytosis. (C) Averaged time courses of endocytosis from scaled responses in control terminals (black, n = 10), and terminals treated with EGTA-AM (red, n = 16) or the AM ester of the pH buffer BCECF [2′,7′-bis-carboxyethyl-5(6)carboxyfluorescein] (blue, n = 6). Bars show SEM at fixed time points. The bold black trace describing the time course of endocytosis in control terminals is a single exponential declining with τ = 1.2 s. The bold red trace describing the time course of endocytosis in terminals treated with EGTA-AM is a double exponential with Afast = 64%, τ fast = 2 s, Aslow = 36%, and τslow = 13.9 s (see text). The peak amplitude of the capacitance responses was 57 ± 8 fF in controls and 50 ± 5 fF in terminals treated with EGTA-AM. (Inset) The averaged Ca2+ currents. (D) Kinetics of endocytosis after the same stimulus delivered in conventional whole-cell recordings with pipette solutions containing 0.5 mM EGTA (blue, n = 6) or 2 mM EGTA (red, n = 10). In 0.5 mM EGTA endocytosis occurred with τ = 1.3 s and so was very similar to that observed in control experiments (the black trace is from D). The decline in membrane area in 2 mM EGTA could be described as a double exponential with Afast = 49%, τ fast = 1.2 s, and τslow = 11.2 s (bold trace). (E) Comparison of averaged responses in 0.4 mM BAPTA (black, n = 15) and 2 mM BAPTA (red, n = 9). In 0.4 mM BAPTA endocytosis occurred with τ = 1.7 s (bold trace) and so was very similar to that observed in control experiments (D). The decline in membrane area in 2 mM BAPTA could be described as a double exponential with Afast = 38%, τ fast = 0.6 s, and τslow = 13 s. (F) The effects of excess EGTA could be reversed. The red trace is a single response measured in the perforated patch configuration after loading the terminal using EGTA-AM. The black trace is a record from the same terminal obtained 2 min after converting to the whole-cell configuration with 0.4 mM BAPTA in the pipette.