DIFFERENTIAL MODULATION OF THE UPTAKE CURRENTS BY REDOX INTERCONVERSION OF CYSTEINE RESIDUES IN THE HUMAN NEURONAL GLUTAMATE TRANSPORTER EAAC1

Control of extrasynaptic glutamate concentration in the central nervou s system is an important determinant of neurotransmission and excitoto xicity, Mechanisms that modulate glutamate transporter function are th erefore critical factors in these processes. The redox modulation of g lutamate uptake was examined by measuring transporter-mediated electri cal currents and radiolabelled amino acid influx in voltage-clamped Xe nopus oocytes expressing the human neuronal glutamate transporter EAAC 1. Up and down changes of the glutamate uptake currents in response to treatment with dithiothreitol and 5,5'-dithio-bis-(2-nitrobenzoic) ac id (DTNB) were observed in oocytes clamped at -60 mV. The redox interc onversion of cysteines induced by dithiothreitol/DTNB influenced the V -max (I-max) of transport, while the apparent affinity for glutamate w as not affected. Formation or breakdown of disulphide groups did not a ffect the pre-steady-state currents, suggesting that these manipulatio ns do not interfere with the Na+ binding/unbinding and/or the charge d istribution on the transporter molecule, The glutamate-evoked net upta ke current of EAAC1 was composed of the inward current from electrogen ic glutamate transport and the current arising from the glutamate-acti vated Cl- conductance. The structural rearrangement produced by the fo rmation or breakdown of disulphide groups only affected the current fr om electrogenic glutamate transport, The electrogenic currents of EAAC 1 were significantly reduced by peroxynitrite, an endogenously occurri ng oxidant formed in certain pathological brain processes, and the mec hanism of inhibition partially depended on the formation of disulphide groups.