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.