Figure 2. Enhanced acid extrusion following activity‐induced cytosolic acid transients follows a time course similar to that of synaptic vesicle membrane endocytosis.

A, representative trace of pH_cyto after a 20 s 40 mM NH₄Cl pulse in MN13‐Ib terminals of w¹¹¹⁸ larvae. B, net molar acid flux (J_H+, mM s^–1) after NH₄Cl as a function of pH_cyto. J_H+ computed from the acid recovery phase (shown in A). Mean ± SEM, n = 6 larvae. C, representative trace of a nerve stimulation‐induced acid transient in MN13‐Ib terminals (40 Hz, 8 s). Grey box highlights post‐stimulus acid efflux. D, average J_H+ every 1 s during the acid recovery phase (observed: recovery phase of pH_cyto transients in C) and predicted intrinsic J_H+ at mean pH_cyto in each 1 s interval (calculated from B). Mean ± SEM, n = 6 larvae. Vertical line indicates time after stimulation at which values of observed and predicted flux are no longer significantly different (P > 0.050, unpaired Student's t test). E, activity‐induced J_H+ (upper panel) following stimulation [calculated as (observed−predicted) acid flux in D]. Mean ± SEM, n = 6 larvae from D. Time course of endocytosis (lower panel, relative fluorescence change in Syn‐pH, average trace of n = 7 larvae). F, activity‐induced [Ca²⁺]_cyto (representative traces) and pH (averaged traces, n = 6 larvae at each frequency) transients from 8 s action potential trains. Light grey box indicates the overlapping pH_cyto range within which J_H+ was calculated. G, quantification of mean J_H+ during stimulation as a function of peak [Ca²⁺]_cyto. Mean ± SEM, n = 6 larvae with values pooled by stimulation frequency and plotted to OGB/AF568 fluorescence ratio, ^* P < 0.050, repeated measures one‐way ANOVA with Holm–Sidak post hoc test. H, τ_rec of acid extrusion phase as a function of peak [Ca²⁺]_cyto. Data plotted as in G; P > 0.050, repeated measures one‐way ANOVA. I, quantification of resting pH_cyto before and after addition of 2 mm Ca²⁺ to Ca²⁺‐free HL3.1. Mean ± SEM. P > 0.050, paired Student's t test, n = 6 larvae. Dashed lines in A–C, E and F are single exponential curves.