The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery

Abstract

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Figures

Figure 1.

Photoinactivation of the a1-I subunit of V0 impairs synaptic transmission. (A) Representation of the V0 a1-I subunit with the Flag (blue) and TC tags in the N- or C-terminal position. (B) Micrograph illustrating the experimental configuration with a patch-clamped presynaptic GFP-expressing neuron (stimulated neuron, colorized in green) and a connected untransfected neuron (voltage clamped to measure the postsynaptic response). Bar, 20 µm. (C) Evolution of the postsynaptic current amplitude in response to presynaptic action potentials (stimulation every 30 s) of rat hippocampal neurons expressing GFP alone (open circles) or GFP + V0 TC-tagged a1-I subunit (filled circles). Current amplitudes were expressed in the percentage of their mean amplitude before the photoinactivating flash. Neurons were successively incubated with 1 µM FlAsH-EDT2 for 10 min and 1 mM BAL without modification of the postsynaptic responses. Photoinactivation was carried for 1 min (gray area). Data shown are from single representative experiments out of three (open circles) or four (filled circles). Typical presynaptic action potentials (Pre) and postsynaptic responses (Post) either before (1) or after (2) the flash are shown. Dotted lines correspond to the mean amplitude of the postsynaptic responses before (top line) or after the photoinactivating flash (bottom line). (D) Averaged effects of V0 a1-I subunit photoinactivation on synaptic transmission. Pooled results include six neuronal pairs, either GABAergic or glutamatergic (paired Student’s t test).

Figure 2.

Photoinactivation of the V0 a1-I or V1 A subunits impairs chromaffin cell catecholamine release in different ways. Chromaffin cells coexpressing either the V0 a1-I or the V1 A subunits bearing a Flag-TC tag and GFP were successively incubated with the FlAsH-EDT2 probe that specifically binds to the TC motif and 1 mM BAL. Photoinactivation was obtained by a 1-min illumination. Catecholamine release was evoked by a 10-s application of 100 mM KCl onto the recorded cell and measured by carbon fiber amperometry. Control release was measured from untransfected cells in the same culture dish or from cells that express GFP alone (both submitted to the FlAsH-EDT2 and BAL treatments and the flash of light). For comparison, catecholamine release from untransfected cells that were only treated by 0.4 µM bafilomycin A1 was measured in parallel. (A) Typical amperometric recordings from a control cell (control); a cell expressing the V0 recombinant a1-I subunit, 5 min after the photoinactivating flash (Flag-TC-a1-I 5′); cells expressing the V1 recombinant A subunit, 5 or 30 min after the flash (Flag-TC-A 5′ and 30’, respectively); and untransfected cells after 5 or 30 min of bafilomycin A1 application (bafilomycin 5′ and 30’, respectively). (B) Number of amperometric spikes detected per cell in the various conditions (means ± SD from 25 to 55 cells). Control cells, 5 or 30 min after the flash (control 5′ and 30’, respectively); cells expressing the V0 a1-I subunit with the Flag-TC tag in N- or C-terminal position, 5 min after the flash (TC-a1-I and a1-I-TC); cells expressing the V1 Flag-TC-A subunit, 5 and 30 min after the flash (TC-A 5′ and TC-A 30’, respectively); and cells bafilomycin A1 treated for 5 or 30 min (bafilo 5′ and 30’). (C) Scheme showing the different parameters of the amperometric response that were measured. A foot current was detected for ∼20–30% of amperometric spikes, regardless the conditions tested. (D) Main characteristics of amperometric spikes in the different conditions tested (as in B). (E) Characteristics of foot currents in the different conditions tested (as in B). Data are means ± SEM (except for B). *, P < 0.05; ***, P < 0.001.

Figure 3.

Manipulation of intragranular pH in PC12 cells. PC12 cells expressing CgA-ECFP were used to measure intragranular pH using FLIM. (A) Calibration curve of fluorescence lifetimes of CgA-ECFP in the function of intragranular pH. PC12 cells were incubated in calibration solutions that contain 10 µM nigericin and 140 mM KCl buffered at the indicated pH. Data are means ± SEM (n = 15–42 cells from six independent experiments). (B) Schemes illustrating how nigericin (N), NH4Cl, or V-ATPase inhibitors affect intragranular pH. (C) Intragranular pH plateau values measured in various conditions. In resting conditions (control [ctrl]) pH was 5.51 ± 0.04 (n = 92 cells). After treatment with 5 µM nigericin (nig), granular pH was 7.15 ± 0.06 (n = 30). Increasing concentrations of NH4Cl led to increasing granular pH, 6.31 ± 0.03 (n = 23) at 5 mM, 6.58 ± 0.04 (n = 37) at 10 mM, and 6.78 ± 0.04 (n = 46) at 20 mM, an effect that was reversible after washing (W) out NH4Cl, pH 5.92 ± 0.05 (n = 20). The V-ATPase inhibitors bafilomycin A1 (B) and saliphenylhalamide A (S) at 0.4 µM raised the pH to 6.80 ± 0.05 (n = 13) and 6.21 ± 0.06 (n = 19), respectively. **, P < 0.01; ***, P < 0.001. (D) Time course of granular pH variations after NH4Cl, bafilomycin A1, or nigericin addition (arrow). Typical experiments are shown that were repeated at least four times.

Figure 4.

Treatments that dissipate the granular pH gradient affect exocytotic release of CgA-EAP from PC12 cells. (A) PC12 cells stably expressing CgA fused with EAP (CgA-EAP) were used to estimate exocytosis of secretory granules induced by KCl depolarization (control [ctrl]). Preincubation with 20 mM NH4Cl for 15 min decreased CgA-EAP release by 52% (NH4), and this was reversed by NH4Cl washout (w). Washing performed in the presence of concanamycin A (w – C) led to partial reversion. (B) Dose dependence of the effect of NH4Cl on CgA-EAP release. Release was 64.2 ± 3.1 (n = 10), 55.2 ± 2.5 (n = 13), and 44.4 ± 2.2% (n = 19) of control release at 5, 10, and 20 mM NH4Cl, respectively. (C) A 15-min preincubation with 5 µM nigericin (nig) markedly reduced CgA-EAP release: 24.4 ± 1.2% (n = 19) of control release. This effect was not reversed after removal of nigericin (washing), 20.8 ± 1.5% (n = 11), unless the K+ ionophore valinomycin (5 µM) was present (washing + valinomycin [V]), a partial reversion that was inhibited by 0.4 µM concanamycin A (washing + valinomycin and concanamycin A [VC]). (D) Effects of 0.4 µM V-ATPase inhibitors. Concanamycin A (C) or bafilomycin A1 (B) did not affect CgA-EAP release, with 99.8 ± 2.5 (n = 25) and 101.1 ± 3.1% (n = 21) of control release, respectively. Saliphenylhalamide A (S) slightly but significantly depressed CgA-EAP release, with 78.5 ± 6.5% (n = 12) of control release. (E) Correlation of CgA-EAP release and intragranular pH in the presence of vehicle (1); reversion of 20 mM NH4Cl (2); 5, 10, and 20 mM NH4Cl (3, 4, 5, respectively); nigericin (6); bafilomycin A1 (B); and saliphenylhalamide A (S). Data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

Incubation with nigericin or 20 mM NH4Cl does not affect exocytosis by impacting Ca2+ influx or calcium internal stores. (A) PC12 cells loaded with Fura-2 were used to estimate variations of the cytosolic Ca2+ concentration. The basal Ca2+ concentration in the cytosol was not affected by the presence of 20 mM NH4Cl. 5 µM nigericin elicited a progressive increase of cytosolic Ca2+ (probably from internal stores because no external Ca2+ was present). K+ depolarization in the presence of 2 mM external calcium induced a marked increase of the cytosolic Ca2+ concentration, which was not much affected by nigericin or NH4Cl. Curves are means from 38 or 39 cells from three to four independent experiments (for clarity, SDs that were always inferior to 10% of the means are not shown). (B) Quantification of the Fura-2 signals resulting from a 5-min-long K+ depolarization, without (control [ctrl]) or in the presence of 5 µM nigericin (nig) or 20 mM NH4Cl (NH4). Means ± SEM from 38 or 39 cells from three to four different cell cultures. (C) NH4Cl (10 and 20 mM) similarly reduced CgA-EAP release induced by KCl depolarization or by the calcium ionophore A23187 (A23187) in the presence of 2 mM external calcium. Data are means ± SEM. (D) PC12 cells expressing CgA-EAP were preincubated 15 min with 10 µM thapsigargin (Thapsi) or with 10 µM calcium ionophore A23187 in the absence of external calcium before triggering CgA-EAP release by KCl. Data were obtained from four (A23187) or three (thapsigargin) different cell cultures. Data are means ± SEM. *, P < 0.05; ***, P < 0.001.

Figure 6.

V-ATPase association acts as a sensor of intragranular pH. (A) CgA-EAP release after a 20-mM NH4Cl incubation (45.9 ± 1.8% of control release, n = 31) was rescued by preincubation (Pre-inc) with 0.4 µM bafilomycin A1 (B; 73.1 ± 3.2%, n = 15) or concanamycin A (C; 66.9 ± 3.2%, n = 16). Protection by 0.4 µM saliphenylhalamide A (S) was less effective (57.6 ± 4.0%, n = 10). (B) CgA-EAP release after 5 µM nigericin (nig) incubation (22.5 ± 1.2% of control release, n = 29) was rescued by preincubation with 0.4 µM bafilomycin A1 (57.5 ± 4.1%, n = 9) or concanamycin A (49.5 ± 2.6%, n = 19). Saliphenylhalamide A was less effective (35.9 ± 4.7%, n = 8). (C) PC12 cells stably expressing CgA-EAP and Flag-TC-a1-I V0 subunit were incubated with 5 µM nigericin (N), 0.4 µM bafilomycin A1, or without treatment (0) for 15 min at 37°C. Cells were incubated with DSP to cross-link the V1–V0 domains before cell lysis and chromaffin granules isolation. The amount of V1 subunit A associated to granules (A) was estimated on Western blots, normalized at equivalent amounts of the endogenous V0 c subunit (c) or Flag a1-I (not depicted). Band intensities were quantified by comparison with increasing amounts of pooled granule fractions (2, 5, and 10). (D) Cells were preincubated for 30 min with 0.4 µM bafilomycin A1 before nigericin treatment (BN) or in physiological medium and then treated with 5 µM nigericin or bafilomycin A1 and treated as in C. (E) Cells treated with 0.4 µM saliphenylhalamide A and as described for D. (F and G) Quantification of the amount of V1 subunit A bound to chromaffin granules, normalized at constant V0 endogenous c subunit (F) or V0 Flag-a1-I subunit (G), expressed in the percentage of bafilomycin A1–treated samples (from three to five different experiments). Data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.