Figure 4.

Granules reseal after exocytosis and reacidify. (A) EGFP–phogrin-labeled granules brighten transiently on exocytosis. (B) Plot of the fluorescence intensity at the granule site along with its time integral (C). (D) Average of 14 events in three cells, obtained as described for Fig. 1C. Continuous line re-plots data with phogrin–DsRed from Fig. 2E. (E–G) Brightening of three granules during application of 50 mM NH₄Cl. The granules in E and F had previously undergone exocytosis, whereas the granule in G had not. (E–G) Plotted as rolling averages of 10 successive measurements to reduce noise. Background was measured as the average of intensities in seven 1.2-μm circles in the image of each cell placed where granules were lacking. (H) Fluorescence changes in cells expressing syntaxin–EGFP on the cell surface. The fluorescence drops markedly when the external medium is acidified (35-μm² membrane area, average of six cells) but changed only slightly when the NH₄Cl-containing solution is applied (six cells). This change may be due to acidic-docked granules that contain small amounts of syntaxin. Because EGFP–phogrin was poorly expressed in our cells, we sought to increase the calcium influx and thus raise the frequency of exocytic events in experiments as desccribed for E and F. This was done by maintaining 50 mM CaCl₂ externally both before (62.5 mM NaCl/3 mM KCl/50 mM CaCl₂/1 mM MgCl₂/10 mM Hepes/10 mM glucose, pH 7.4, 300 milliosmolal) and during (105 mM KCl/50 mM CaCl₂/0.7 mM MgCl₂/1 mM NaH₂PO₄/10 mM Hepes, 379 milliosmolal) stimulation. The 50 mM [Ca²⁺] did not significantly change the fluorescence response during exocytosis in EGFP–phogrin-expressing cells. Fluorescence rose abruptly by a factor 5.5 ± 0.8 and declined with a half-time of 7.9 ± 0.9 s (16 events, five cells).