Ca2+-induced deprotonation of peptide hormones inside secretory vesicles in preparation for release

Abstract

The acidic environment inside secretory vesicles ensures that neuropeptides and peptide hormones are packaged in a concentrated condensed form. Although this is optimal for storage, decondensation limits release. Thus, it would be advantageous to alter the physical state of peptides in preparation for exocytosis. Here, we report that depolarization of the plasma membrane rapidly increases enhanced green fluorescent protein (EGFP)-tagged hormone fluorescence inside secretory vesicles. This effect requires Ca2+ influx and persists when exocytosis is inhibited by N-ethylmaleimide. Peptide deprotonation appears to produce this response, because it is not seen when the vesicle pH gradient is collapsed or when a pH-insensitive GFP variant is used. These data demonstrate that Ca2+ evokes alkalinization of the inside of secretory vesicles before exocytosis. Thus, Ca2+ influx into the cytoplasm alters the physical state of intravesicular contents in preparation for release.

Fig. 1.

Depolarization-induced proANF-EGFP fusion protein release is accompanied by an increase in fluorescence. InA and B, cells were superfused first in normal saline and then switched to 100 mmK⁺ saline (arrows). The fluorescence decrease, indicative of peptide release, was preceded by either a delay (A) or an increase (B) in fluorescence. C, A double-pulse depolarization stimulus (S, arrow), from a holding potential of −80 to 10 mV for 500 msec with a 5 sec interpulse, was delivered to the cell via a patch pipette in whole-cell configuration. Note the the increase in fluorescence was evident immediately after the double pulse. D, A similar fluorescence increase was observed in response to the double-pulse stimulus (S,arrow), without a net fluorescence decrease.

Fig. 2.

The depolarization-induced fluorescence change does not depend on exocytosis but requires extracellular Ca²⁺. Cells were pretreated with 0.2 mmNEM to block peptide release. A, Cells were superfused with normal saline and then with 100 mmK⁺ saline (bar). Note the apparent and uncontaminated increase in fluorescence. B, Cells were superfused with Ca²⁺-free normal saline, then with Ca²⁺-free 100 mmK⁺ saline, and finally with 100 mmK⁺ saline containing 5 mmCa²⁺. The fluorescence increase induced by 100 mm K⁺ saline occurred only when Ca²⁺ was present. C, Cd²⁺ prevented fluorescence increase caused by Ca²⁺. Cells were superfused with normal saline containing 0.2 mm Cd²⁺ and then with 100 mm K⁺ saline containing 0.2 mm Cd²⁺. D, Ba²⁺ mimicked Ca²⁺ in causing the fluorescence increase. Cells were initially superfused with normal saline and then with Ca²⁺-free Ba²⁺ containing 100 mmK⁺ saline. E, Quantification of divalent dependence of the effect of depolarization after NEM treatment. n = 4, 4, 6, and 4 for 0 Ca²⁺, 5 mm Ca²⁺, 5 mm Ca²⁺ plus 0.2 mmCd²⁺, and 5 mm Ba²⁺, respectively.

Fig. 3.

Fluorescence of EGFP-tagged peptide hormone is sensitive to pH. A, Fluorescence excitation spectra of proANF-EGFP fusion protein at two different pH values.B, Microfluorimetric recordings of the responses of proANF-EGFP fusion protein expressed in cells to solutions at various pH levels. The response of the fluorescence of proANF-EGFP to changing pH was quick and reversible. C, Titration curve for the relative fluorescence of proANF-EGFP.

Fig. 4.

The depolarization-induced increase in intravesicular fluorescence requires a pH gradient. Cells were initially superfused with normal saline containing a pH-collapsing agent [1 μm monensin (A), 1 μm nigericin (B), or 1 μm FCCP (C)] and then with 100 mm K⁺ saline containing the same pH-collapsing agent. Note that fluorescence decrease occurred almost immediately after 100 mmK⁺ application. D, Comparison of delay from the application of 100 mm K⁺to the onset of fluorescence decrease in cells without pretreatment (C) and those pretreated with monensin (M), nigericin (N), or FCCP (F). n = 11, 8, 5, and 5 for control, monensin, nigericin, and FCCP, respectively. *p < 0.01 versus control.

Fig. 5.

Fluorescence of proANF-Sapphire fusion protein is insensitive to pH. A, Fluorescence excitation spectra of proANF-Sapphire GFP fusion protein at two different pH values.B, Microfluorimetric recordings of the responses of proANF-Sapphire fusion protein to bath superfusion of normal saline at various pH levels. C, Titration curve for the relative fluorescence of Sapphire-tagged proANF.

Fig. 6.

The apparent delay after application of 100 mm K⁺ saline is no longer observed when using proANF-Sapphire. A, A cell was initially superfused with normal saline and then with 100 mmK⁺ saline. Note that fluorescence decrease occurred almost immediately after 100 mm K⁺application. Also, no increase in fluorescence was observed with proANF-Sapphire. B, Comparison of delay from the application of 100 mm K⁺ saline to the onset of fluorescence decrease in cells transfected with proANF-EGFP (EGFP) and those transfected with proANF-Sapphire (Sph). n = 11 and 12 for proANF-EGFP and proANF-Sapphire, respectively. *p < 0.01 versus EGFP.

Fig. 7.

NEM increases fluorescence in cells that are transfected with proANF-EGFP but not those with proANF-Sapphire.A, A cell expressing proANF-EGFP was initially superfused with normal saline, then with normal saline containing 0.2 mm NEM, and finally with normal saline containing 1 μm monensin. Note that fluorescence increased gradually after the start of NEM superfusion and that fluorescence increased very quickly when superfusion was switched to monensin (M). B, A cell expressing proANF-Sapphire was initially superfused with normal saline and then with normal saline containing 0.2 mm NEM. NEM did not have any effect on fluorescence of proANF-Sapphire. C, A proANF-Sapphire-expressing cell was pretreated with NEM and DTT. After initial superfusion with normal saline, the solution was switched to normal saline containing 1 μm monensin.