REGULATION OF GLUTAMATE TRANSPORT INTO SYNAPTIC VESICLES BY CHLORIDE AND PROTON GRADIENT

Glutamate uptake into synaptic vesicles is driven by an electrochemica l proton gradient formed across the membrane by a vacuolar H+-ATPase. Chloride has a biphasic effect on glutamate transport, which it activa tes at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in th e V-max of transport, whereas inhibition by high chloride concentratio ns was related to an increase in K-m to glutamate. Both stimulation an d inhibition by Cl- were observed in the presence of A23187 or (NH4)(2 )SO4, two substances that dissipate the proton gradient (Delta pH). Wi th the use of these agents, we show that the transmembrane potential r egulates the apparent affinity for glutamate, whereas the Delta pH ant agonizes the effect of high chloride concentrations and is important f or retaining glutamate inside the vesicles. Selective dissipation of D elta pH in the presence of chloride led to a significant glutamate eff lux from the vesicles and promoted a decrease in the velocity of gluta mate uptake. The H+-ATPase activity was stimulated when the Delta pH c omponent was dissipated. Glutamate efflux induced by chloride was satu rable, and half-maximal effect was attained in the presence of 30 mM C l-. The results indicate that: (i) both transmembrane potential and De lta pH modulate the glutamate uptake at different levels and (ii) chlo ride affects glutamate transport by two different mechanisms. One is r elated to a change of the proportions between the transmembrane potent ial and the Delta pH components of the electrochemical proton gradient , and the other involves a direct interaction of the anion with the gl utamate transporter.