Regulation of glutamate transport into synaptic vesicles by chloride and proton gradient.
Glutamate uptake into synaptic vesicles is driven by an electrochemical proton gradient formed across the membrane by a vacuolar H+-ATPase. Chloride has a biphasic effect on glutamate transport, which it activates at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in the Vmax of transport, whereas inhibition by high chloride concentrations was related to an increase in Km to glutamate. Both stimulation and inhibition by Cl- were observed in the presence of A23187 or (NH4)2SO4, two substances that dissipate the proton gradient (deltapH). With the use of these agents, we show that the transmembrane potential regulates the apparent affinity for glutamate, whereas the deltapH antagonizes the effect of high chloride concentrations and is important for retaining glutamate inside the vesicles. Selective dissipation of deltapH in the presence of chloride led to a significant glutamate efflux from the vesicles and promoted a decrease in the velocity of glutamate uptake. The H+-ATPase activity was stimulated when the deltapH component was dissipated. Glutamate efflux induced by chloride was saturable, and half-maximal effect was attained in the presence of 30 mM Cl-. The results indicate that: (i) both transmembrane potential and deltapH modulate the glutamate uptake at different levels and (ii) chloride affects glutamate transport by two different mechanisms. One is related to a change of the proportions between the transmembrane potential and the deltapH components of the electrochemical proton gradient, and the other involves a direct interaction of the anion with the glutamate transporter.
Wolosker
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Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Cidade Universitária, Brazil.
de Souza
D O
DO
de Meis
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D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*
D002712:Chlorides; D000001:Calcimycin; D018698:Glutamic Acid; D000645:Ammonium Sulfate