Energy coupling of L-glutamate transport and vacuolar H⁺-ATPf in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require Cl^-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K⁺. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K⁺. Like formation of a membrane potential, ATP-dependent L-[³H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K⁺. The L- [³H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H⁺ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H⁺. These results were consistent with the notion that the vacuolar H⁺-ATPase of synaptic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl^-, glutamate or nigericin, indicating that an electrochemical H⁺ gradient had no effect on the ATPase activity.