Hydrogen peroxide inhibits the vacuolar H+-ATPase in brain synaptic vesicles at micromolar concentrations

Hydrogen peroxide (H2O2) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H2O2 on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H+-ATPase (V-ATPase) in the vesicle membrane. We report here that the VATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H2O2. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H2O2-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H2O2. These and other results suggest that the mechanism of inhibition of the V-ATPase by H2O2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.