ATP-dependent proton translocation across the rat brain synaptic vesicle membrane

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Abstract
Changes in pH in rat brain synaptic vesicles (SV) were studied with the fluorescent slightly basic dye, acridine orange. The pH value in isolated SV was acidic, which was confirmed by the ionophore sensitive accumulation of the dye. Addition of ATP provoked further acidification of the intravesicular medium. The acidification rate reached a maximum after dissipation of the existing H+ gradient seen during preincubation in the absence of ATP. The ATP-dependent acidification was eliminated by the protonophore carbonylcyanide m-chlorophenylhydrazone, H4Cl or the detergent triton X-199 (0.025%). Valinomycin inhibited the ATP-dependent translocation of H+ whatever the incubation medium (with KCl or NaCl). Dicyclohexylcarbodiimide, a known inhibitor of proton ATPases (100 .mu.M) as well as ethylmaleimide (100 .mu.M) completely blocked H+ translocation whereas oligomycin, a specific blocker of mitochondrial H+-ATPase, and ouabain did not influence that process. ATP induced H+ translocation only in the presence of Mn2+ or Mg2+ but not in the presence of Ca2+. The translocation of H+ was not affected by the replacement of univalent cations (KCl, NaCl or Cl), however, it was prevented completely upon replacement of the penetrating anion Cl- by the nonpenetrating anion O42- or upon replacement of the salts by sucrose. The ATP-dependent translocation of H+ in SV is mediated via H+-ATPase which maintains the low pH value in SV.